Nanoparticles having oligonucleotides attached thereto and uses therefor

ABSTRACT

The invention provides methods of detecting a nucleic acid. The methods comprise contacting the nucleic acid with one or more types of particles having oligonucleotides attached thereto. In one embodiment of the method, the oligonucleotides are attached to nanoparticles and have sequences complementary to portions of the sequence of the nucleic acid. A detectable change (preferably a color change) is brought about as a result of the hybridization of the oligonucleotides on the nanoparticles to the nucleic acid. The invention also provides compositions and kits comprising particles. The invention further provides methods of synthesizing unique nanoparticle-oligonucleotide conjugates, the conjugates produced by the methods, and methods of using the conjugates. In addition, the invention provides nanomaterials and nanostructures comprising nanoparticles and methods of nanofabrication utilizing nanoparticles. Finally, the invention provides a method of separating a selected nucleic acid from other nucleic acids.

This application is a continuation of U.S. application Ser. No.09/603,830 filed Jun. 26, 2000, now U.S. Pat. No. 6,506,564, issued Jan.14, 2003, which is a continuation-in-part of application Ser. No.09/344,667, filed Jun. 25, 1999, now U.S. Pat. No. 6,361,944, issuedMar. 26, 2002, which was a continuation-in-part of application Ser. No.09/240,755, filed Jan. 29, 1999 (abandoned), which was acontinuation-in-part of pending PCT application PCT/US97/12783, whichwas filed Jul. 21, 1997. Benefit of provisional applications No.60/031,809, filed Jul. 29, 1996, and 60/200,161, filed Apr. 26, 2000 isalso hereby claimed.

This invention was made with government support under NationalInstitutes Of Health (NIH) grant GM10265 and Army Research Office (ARO)grant DAAG55-0967-1-0133. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The invention relates to methods of detecting nucleic acids, whethernatural or synthetic, and whether modified or unmodified. The inventionalso relates to materials for detecting nucleic acids and methods ofmaking those materials. The invention further relates to methods ofnanofabrication. Finally, the invention relates to methods of separatinga selected nucleic acid from other nucleic acids.

BACKGROUND OF THE INVENTION

The development of methods for detecting and sequencing nucleic acids iscritical to the diagnosis of genetic, bacterial, and viral diseases. SeeMansfield, E. S. et al. Molecular and Cellular Probes, 9, 145–156(1995). At present, there are a variety of methods used for detectingspecific nucleic acid sequences. Id. However, these methods arecomplicated, time-consuming and/or require the use of specialized andexpensive equipment. A simple, fast method of detecting nucleic acidswhich does not require the use of such equipment would clearly bedesirable.

A variety of methods have been developed for assembling metal andsemiconductor colloids into nanomaterials. These methods have focused onthe use of covalent linker molecules that possess functionalities atopposing ends with chemical affinities for the colloids of interest. Oneof the most successful approaches to date, Brust et al., Adv. Mater., 7,795–797 (1995), involves the use of gold colloids and well-establishedthiol adsorption chemistry, Bain & Whitesides, Angew. Chem. Int. Ed.Engl., 28, 506–512 (1989) and Dubois & Nuzzo, Annu. Rev. Phys. Chem.,43, 437–464 (1992). In this approach, linear alkanedithiols are used asthe particle linker molecules. The thiol groups at each end of thelinker molecule covalently attach themselves to the colloidal particlesto form aggregate structures. The drawbacks of this method are that theprocess is difficult to control and the assemblies are formedirreversibly. Methods for systematically controlling the assemblyprocess are needed if the materials properties of these structures areto be exploited fully.

The potential utility of DNA for the preparation of biomaterials and innanofabrication methods has been recognized. In this work, researchershave focused on using the sequence-specific molecular recognitionproperties of oligonucleotides to design impressive structures withwell-defined geometric shapes and sizes. Shekhtman et al., New J. Chem.,17, 757–763 (1993); Shaw & Wang, Science, 260, 533–536 (1993); Chen etal., J. Am Chem. Soc., 111, 6402–6407 (1989); Chen & Seeman, Nature,350, 631–633 (1991); Smith and Feigon, Nature, 356, 164–168 (1992); Wanget al., Biochem., 32, 1899–1904 (1993); Chen et al., Biochem., 33,13540–13546 (1994); Marsh et al., Nucleic Acids Res., 23, 696–700(1995); Mirkin, Annu. Review Biophys. Biomol. Struct., 23,541–576(1994); Wells, J. Biol. Chem., 263, 1095–1098 (1988); Wang et al.,Biochem., 30, 5667–5674 (1991). However, the theory of producing DNAstructures is well ahead of experimental confirmation. Seeman et al.,New J. Chem., 17, 739–755 (1993).

SUMMARY OF THE INVENTION

The invention provides methods of detecting nucleic acids. In oneembodiment, the method comprises contacting a nucleic acid with a typeof nanoparticles having oligonucleotides attached thereto(nanoparticle-oligonucleotide conjugates). The nucleic acid has at leasttwo portions, and the oligonucleotides on each nanoparticle have asequence complementary to the sequences of at least two portions of thenucleic acid. The contacting takes place under conditions effective toallow hybridization of the oligonucleotides on the nanoparticles withthe nucleic acid. The hybridization of the oligonucleotides on thenanoparticles with the nucleic acid results in a detectable change.

In another embodiment, the method comprises contacting a nucleic acidwith at least two types of nanoparticles having oligonucleotidesattached thereto. The oligonucleotides on the first type ofnanoparticles have a sequence complementary to a first portion of thesequence of the nucleic acid. The oligonucleotides on the second type ofnanoparticles have a sequence complementary to a second portion of thesequence of the nucleic acid. The contacting takes place underconditions effective to allow hybridization of the oligonucleotides onthe nanoparticles with the nucleic acid, and a detectable change broughtabout by this hybridization is observed.

In a further embodiment, the method comprises providing a substratehaving a first type of nanoparticles attached thereto. The first type ofnanoparticles has oligonucleotides attached thereto, and theoligonucleotides have a sequence complementary to a first portion of thesequence of a nucleic acid. The substrate is contacted with the nucleicacid under conditions effective to allow hybridization of theoligonucleotides on the nanoparticles with the nucleic acid. Then, asecond type of nanoparticles having oligonucleotides attached thereto isprovided. The oligonucleotides have a sequence complementary to one ormore other portions of the sequence of the nucleic acid, and the nucleicacid bound to the substrate is contacted with the second type ofnanoparticle-oligonucleotide conjugates under conditions effective toallow hybridization of the oligonucleotides on the second type ofnanoparticles with the nucleic acid. A detectable change may beobservable at this point. The method may further comprise providing abinding oligonucleotide having a selected sequence having at least twoportions, the first portion being complementary to at least a portion ofthe sequence of the oligonucleotides on the second type ofnanoparticles. The binding oligonucleotide is contacted with the secondtype of nanoparticle-oligonucleotide conjugates bound to the substrateunder conditions effective to allow hybridization of the bindingoligonucleotide to the oligonucleotides on the nanoparticles. Then, athird type of nanoparticles having oligonucleotides attached thereto,the oligonucleotides having a sequence complementary to the sequence ofa second portion of the binding oligonucleotide, is contacted with thebinding oligonucleotide bound to the substrate under conditionseffective to allow hybridization of the binding oligonucleotide to theoligonucleotides on the nanoparticles. Finally, the detectable changeproduced by these hybridizations is observed.

In yet another embodiment, the method comprises contacting a nucleicacid with a substrate having oligonucleotides attached thereto, theoligonucleotides having a sequence complementary to a first portion ofthe sequence of the nucleic acid. The contacting takes place underconditions effective to allow hybridization of the oligonucleotides onthe substrate with the nucleic acid. Then, the nucleic acid bound to thesubstrate is contacted with a first type of nanoparticles havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to a second portion of the sequence of thenucleic acid. The contacting takes place under conditions effective toallow hybridization of the oligonucleotides on the nanoparticles withthe nucleic acid. Next, the first type of nanoparticle-oligonucleotideconjugates bound to the substrate is contacted with a second type ofnanoparticles having oligonucleotides attached thereto, theoligonucleotides on the second type of nanoparticles having a sequencecomplementary to at least a portion of the sequence of theoligonucleotides on the first type of nanoparticles, the contactingtaking place under conditions effective to allow hybridization of theoligonucleotides on the first and second types of nanoparticles.Finally, a detectable change produced by these hybridizations isobserved.

In another embodiment, the method comprises contacting a nucleic acidwith a substrate having oligonucleotides attached thereto, theoligonucleotides having a sequence complementary to a first portion ofthe sequence of the nucleic acid. The contacting takes place underconditions effective to allow hybridization of the oligonucleotides onthe substrate with the nucleic acid. Then, the nucleic acid bound to thesubstrate is contacted with liposomes having oligonucleotides attachedthereto, the oligonucleotides having a sequence complementary to aportion of the sequence of the nucleic acid. This contacting takes placeunder conditions effective to allow hybridization of theoligonucleotides on the liposomes with the nucleic acid. Next, theliposome-oligonucleotide conjugates bound to the substrate are contactedwith a first type of nanoparticles having at least a first type ofoligonucleotides attached thereto. The first type of oligonucleotideshave a hydrophobic group attached to the end not attached to thenanoparticles, and the contacting takes place under conditions effectiveto allow attachment of the oligonucleotides on the nanoparticles to theliposomes as a result of hydrophobic interactions. A detectable changemay be observable at this point. The method may further comprisecontacting the first type of nanoparticle-oligonucleotide conjugatesbound to the liposomes with a second type of nanoparticles havingoligonucleotides attached thereto. The first type of nanoparticles havea second type of oligonucleotides attached thereto which have a sequencecomplementary to at least a portion of the sequence of theoligonucleotides on the second type of nanoparticles, and theoligonucleotides on the second type of nanoparticles having a sequencecomplementary to at least a portion of the sequence of the second typeof oligonucleotides on the first type of nanoparticles. The contactingtakes place under conditions effective to allow hybridization of theoligonucleotides on the first and second types of nanoparticles. Then, adetectable change is observed.

In another embodiment, the method comprises contacting a nucleic acid tobe detected with a substrate having oligonucleotides attached thereto.The oligonucleotides have a sequence complementary to a first portion ofthe sequence of said nucleic acid, the contacting takes place underconditions effective to allow hybridization of the oligonucleotides onthe substrate with said nucleic acid. Next, said nucleic acid bound tothe substrate is contacted with a type of nanoparticles havingoligonucleotides attached thereto. The oligonucleotides have a sequencecomplementary to a second portion of the sequence of said nucleic acid.The contacting takes place under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles with saidnucleic acid. Then, the substrate is contacted with silver stain toproduce a detectable change, and the detectable change is observed.

In yet another embodiment, the method comprises providing a substratehaving a first type of nanoparticles attached thereto. The nanoparticleshave oligonucleotides attached thereto, the oligonucleotides having asequence complementary to a first portion of the sequence of a nucleicacid to be detected. Then, the nucleic acid is contacted with thenanoparticles attached to the substrate under conditions effective toallow hybridization of the oligonucleotides on the nanoparticles withsaid nucleic acid. Next, an aggregate probe comprising at least twotypes of nanoparticles having oligonucleotides attached thereto isprovided. The nanoparticles of the aggregate probe are bound to eachother as a result of the hybridization of some of the oligonucleotidesattached to them. At least one of the types of nanoparticles of theaggregate probe have oligonucleotides attached thereto which have asequence complementary to a second portion of the sequence of saidnucleic acid. Finally, said nucleic acid bound to the substrate iscontacted with the aggregate probe under conditions effective to allowhybridization of the oligonucleotides on the aggregate probe with saidnucleic acid, and a detectable change is observed.

In a further embodiment, the method comprises providing a substratehaving oligonucleotides attached thereto. The oligonucleotides have asequence complementary to a first portion of the sequence of a nucleicacid to be detected. An aggregate probe comprising at least two types ofnanoparticles having oligonucleotides attached thereto is provided. Thenanoparticles of the aggregate probe are bound to each other as a resultof the hybridization of some of the oligonucleotides attached to them.At least one of the types of nanoparticles of the aggregate probe haveoligonucleotides attached thereto which have a sequence complementary toa second portion of the sequence of said nucleic acid. The nucleic acid,the substrate and the aggregate probe are contacted under conditionseffective to allow hybridization of said nucleic acid with theoligonucleotides on the aggregate probe and with the oligonucleotides onthe substrate, and a detectable change is observed.

In a further embodiment, the method comprises providing a substratehaving oligonucleotides attached thereto. An aggregate probe comprisingat least two types of nanoparticles having oligonucleotides attachedthereto is provided. The nanoparticles of the aggregate probe are boundto each other as a result of the hybridization of some of theoligonucleotides attached to them. At least one of the types ofnanoparticles of the aggregate probe have oligonucleotides attachedthereto which have a sequence complementary to a first portion of thesequence of a nucleic acid to be detected. A type of nanoparticleshaving at least two types of oligonucleotides attached thereto isprovided. The first type of oligonucleotides has a sequencecomplementary to a second portion of the sequence of said nucleic acid,and the second type of oligonucleotides has a sequence complementary toat least a portion of the sequence of the oligonucleotides attached tothe substrate. The nucleic acid, the aggregate probe, the nanoparticlesand the substrate are contacted under conditions effective to allowhybridization of said nucleic acid with the oligonucleotides on theaggregate probe and on the nanoparticles and hybridization of theoligonucleotides on the nanoparticles with the oligonucleotides on thesubstrate, and a detectable change is observed.

In another embodiment, the method comprises contacting a nucleic acid tobe detected with a substrate having oligonucleotides attached thereto.The oligonucleotides have a sequence complementary to a first portion ofthe sequence of said nucleic acid. The contacting takes place underconditions effective to allow hybridization of the oligonucleotides onthe substrate with said nucleic acid. The nucleic acid bound to thesubstrate is contacted with liposomes having oligonucleotides attachedthereto, the oligonucleotides having a sequence complementary to aportion of the sequence of said nucleic acid. The contacting takes placeunder conditions effective to allow hybridization of theoligonucleotides on the liposomes with said nucleic acid. An aggregateprobe comprising at least two types of nanoparticles havingoligonucleotides attached thereto is provided. The nanoparticles of theaggregate probe are bound to each other as a result of the hybridizationof some of the oligonucleotides attached to them, at least one of thetypes of nanoparticles of the aggregate probe having oligonucleotidesattached thereto which have a hydrophobic group attached to the end notattached to the nanoparticles. The liposomes bound to the substrate arecontacted with the aggregate probe under conditions effective to allowattachment of the oligonucleotides on the aggregate probe to theliposomes as a result of hydrophobic interactions, and a detectablechange is observed.

In yet another embodiment, the method comprises providing a substratehaving oligonucleotides attached thereto. The oligonucleotides having asequence complementary to a first portion of the sequence of a nucleicacid to be detected. A core probe comprising at least two types ofnanoparticles is provided. Each type of nanoparticles hasoligonucleotides attached thereto which are complementary to theoligonucleotides on at least one of the other types of nanoparticles.The nanoparticles of the aggregate probe are bound to each other as aresult of the hybridization of the oligonucleotides attached to them.Next, a type of nanoparticles having two types of oligonucleotidesattached thereto is provided. The first type of oligonucleotides has asequence complementary to a second portion of the sequence of saidnucleic acid, and the second type of oligonucleotides has a sequencecomplementary to a portion of the sequence of the oligonucleotidesattached to at least one of the types of nanoparticles of the coreprobe. The nucleic acid, the nanoparticles, the substrate and the coreprobe are contacted under conditions effective to allow hybridization ofsaid nucleic acid with the oligonucleotides on the nanoparticles andwith the oligonucleotides on the substrate and to allow hybridization ofthe oligonucleotides on the nanoparticles with the oligonucleotides onthe core probe, and a detectable change is observed.

Another embodiment of the method comprises providing a substrate havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to a first portion of the sequence of a nucleicacid to be detected. A core probe comprising at least two types ofnanoparticles is provided. Each type of nanoparticles hasoligonucleotides attached thereto which are complementary to theoligonucleotides on at least one other type of nanoparticles. Thenanoparticles of the aggregate probe are bound to each other as a resultof the hybridization of the oligonucleotides attached to them. A type oflinking oligonucleotides comprising a sequence complementary to a secondportion of the sequence of said nucleic acid and a sequencecomplementary to a portion of the sequence of the oligonucleotidesattached to at least one of the types of nanoparticles of the core probeis provided. The nucleic acid, the linking oligonucleotides, thesubstrate and the core probe are contacted under conditions effective toallow hybridization of said nucleic acid with the linkingoligonucleotides and with the oligonucleotides on the substrate and toallow hybridization of the oligonucleotides on the linkingoligonucleotides with the oligonucleotides on the core probe, and adetectable change is observed.

In yet another embodiment, the method comprises providing nanoparticleshaving oligonucleotides attached thereto and providing one or more typesof binding oligonucleotides. Each of the binding oligonucleotides hastwo portions. The sequence of one portion is complementary to thesequence of one of the portions of the nucleic acid, and the sequence ofthe other portion is complementary to the sequence of theoligonucleotides on the nanoparticles. The nanoparticle-oligonucleotideconjugates and the binding oligonucleotides are contacted underconditions effective to allow hybridization of the oligonucleotides onthe nanoparticles with the binding oligonucleotides. The nucleic acidand the binding oligonucleotides are contacted under conditionseffective to allow hybridization of the binding oligonucleotides withthe nucleic acid. Then, a detectable change is observed. Thenanoparticle-oligonucleotide conjugates may be contacted with thebinding oligonucleotides prior to being contacted with the nucleic acid,or all three may be contacted simultaneously.

In another embodiment, the method comprises contacting a nucleic acidwith at least two types of particles having oligonucleotides attachedthereto. The oligonucleotides on the first type of particles have asequence complementary to a first portion of the sequence of the nucleicacid and have energy donor molecules on the ends not attached to theparticles. The oligonucleotides on the second type of particles have asequence complementary to a second portion of the sequence of thenucleic acid and have energy acceptor molecules on the ends not attachedto the particles. The contacting takes place under conditions effectiveto allow hybridization of the oligonucleotides on the particles with thenucleic acid, and a detectable change brought about by thishybridization is observed. The energy donor and acceptor molecules maybe fluorescent molecules.

In a further embodiment, the method comprises providing a type ofmicrospheres having oligonucleotides attached thereto. Theoligonucleotides have a sequence complementary to a first portion of thesequence of the nucleic acid and are labeled with a fluorescentmolecule. A type of nanoparticles having oligonucleotides attachedthereto and which produce a detectable change is also provided. Theseoligonucleotides have a sequence complementary to a second portion ofthe sequence of the nucleic acid. The nucleic acid is contacted with themicrospheres and the nanoparticles under conditions effective to allowhybridization of the oligonucleotides on the latex microspheres and onthe nanoparticles with the nucleic acid. Then, changes in fluorescence,another detectable change, or both are observed.

In another embodiment, the method comprises providing a first type ofmetallic or semiconductor nanoparticles having oligonucleotides attachedthereto. The oligonucleotides have a sequence complementary to a firstportion of the sequence of the nucleic acid and are labeled with afluorescent molecule. A second type of metallic or semiconductornanoparticles having oligonucleotides attached thereto is also provided.These oligonucleotides have a sequence complementary to a second portionof the sequence of the nucleic acid and are also labeled with afluorescent molecule. The nucleic acid is contacted with the two typesof nanoparticles under conditions effective to allow hybridization ofthe oligonucleotides on the two types of nanoparticles with the nucleicacid. Then, changes in fluorescence are observed.

In a further embodiment, the method comprises providing a type ofparticle having oligonucleotides attached thereto. The oligonucleotideshave a first portion and a second portion, both portions beingcomplementary to portions of the sequence of the nucleic acid. A type ofprobe oligonucleotides comprising a first portion and a second portionis also provided. The first portion has a sequence complementary to thefirst portion of the oligonucleotides attached to the particles, andboth portions are complementary to portions of the sequence of thenucleic acid. The probe oligonucleotides are also labeled with areporter molecule at one end. Then, the particles and the probeoligonucleotides are contacted under conditions effective to allow forhybridization of the oligonucleotides on the particles with the probeoligonucleotides to produce a satellite probe. Then, the satellite probeis contacted with the nucleic acid under conditions effective to providefor hybridization of the nucleic acid with the probe oligonucleotides.The particles are removed and the reporter molecule detected.

In yet another embodiment of the method of the invention, a nucleic acidis detected by contacting the nucleic acid with a substrate havingoligonucleotides attached thereto. The oligonucleotides have a sequencecomplementary to a first portion of the sequence of the nucleic acid.The oligonucleotides are located between a pair of electrodes located onthe substrate. The contacting takes place under conditions effective toallow hybridization of the oligonucleotides on the substrate with thenucleic acid. Then, the nucleic acid bound to the substrate, iscontacted with a type of nanoparticles. The nanoparticles are made of amaterial which can conduct electricity. The nanoparticles will have oneor more types of oligonucleotides attached to them, at least one of thetypes of oligonucleotides having a sequence complementary to a secondportion of the sequence of the nucleic acid. The contacting takes placeunder conditions effective to allow hybridization of theoligonucleotides on the nanoparticles with the nucleic acid. If thenucleic acid is present, a change in conductivity can be detected. In apreferred embodiment, the substrate will have a plurality of pairs ofelectrodes located on it in an array to allow for the detection ofmultiple portions of a single nucleic acid, the detection of multipledifferent nucleic acids, or both. Each of the pairs of electrodes in thearray will have a type of oligonucleotides attached to the substratebetween the two electrodes.

The invention further provides a method of detecting a nucleic acidwherein the method is performed on a substrate. The method comprisesdetecting the presence, quantity or both, of the nucleic acid with anoptical scanner.

The invention further provides kits for detecting nucleic acids. In oneembodiment, the kit comprises at least one containers the containerholding at least two types of nanoparticles having oligonucleotidesattached thereto. The oligonucleotides on the first type ofnanoparticles have a sequence complementary to the sequence of a firstportion of a nucleic acid. The oligonucleotides on the second type ofnanoparticles have a sequence complementary to the sequence of a secondportion of the nucleic acid.

Alternatively, the kit may comprise at least two containers. The firstcontainer holds nanoparticles having oligonucleotides attached theretowhich have a sequence complementary to the sequence of a first portionof a nucleic acid. The second container holds nanoparticles havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a second portion of the nucleic acid.

In a further embodiment, the kit comprises at least one container. Thecontainer holds metallic or semiconductor nanoparticles havingoligonucleotides attached thereto. The oligonucleotides have a sequencecomplementary to portion of a nucleic acid and have fluorescentmolecules attached to the ends of the oligonucleotides not attached tothe nanoparticles.

In yet another embodiment, the kit comprises a substrate, the substratehaving attached thereto nanoparticles, the nanoparticles havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid. The kit also includesa first container holding nanoparticles having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a secondportion of the nucleic acid. The kit further includes a second containerholding a binding oligonucleotide having a selected sequence having atleast two portions, the first portion being complementary to at least aportion of the sequence of the oligonucleotides on the nanoparticles inthe first container. The kit also includes a third container holdingnanoparticles having oligonucleotides attached thereto, theoligonucleotides having a sequence complementary to the sequence of asecond portion of the binding oligonucleotide.

In another embodiment, the kit comprises a substrate havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid, a first containerholding nanoparticles having oligonucleotides attached thereto whichhave a sequence complementary to the sequence of a second portion of thenucleic acid, and a second container holding nanoparticles havingoligonucleotides attached thereto which have a sequence complementary toat least a portion of the oligonucleotides attached to the nanoparticlesin the first container.

In yet another embodiment, the kit comprises a substrate, a firstcontainer holding nanoparticles, a second container holding a first typeof oligonucleotides having a sequence complementary to the sequence of afirst portion of a nucleic acid, a third container holding a second typeof oligonucleotides having a sequence complementary to the sequence of asecond portion of the nucleic acid, and a fourth container holding athird type of oligonucleotides having a sequence complementary to atleast a portion of the sequence of the second type of oligonucleotides.

In a further embodiment, the kit comprises a substrate havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid. The kit also includesa first container holding liposomes having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a secondportion of the nucleic acid and a second container holding nanoparticleshaving at least a first type of oligonucleotides attached thereto, thefirst type of oligonucleotides having a hydrophobic group attached tothe end not attached to the nanoparticles so that the nanoparticles canbe attached to the liposomes by hydrophobic interactions. The kit mayfurther comprise a third container holding a second type ofnanoparticles having oligonucleotides attached thereto, theoligonucleotides having a sequence complementary to at least a portionof the sequence of a second type of oligonucleotides attached to thefirst type of nanoparticles. The second type of oligonucleotidesattached to the first type of nanoparticles have a sequencecomplementary to the sequence of the oligonucleotides on the second typeof nanoparticles.

In another embodiment, the kit comprises a substrate havingnanoparticles attached to it. The nanoparticles have oligonucleotidesattached to them which have a sequence complementary to the sequence ofa first portion of a nucleic acid. The kit also includes a firstcontainer holding an aggregate probe. The aggregated probe comprises atleast two types of nanoparticles having oligonucleotides attached tothem. The nanoparticles of the aggregate probe are bound to each otheras a result of the hybridization of some of the oligonucleotidesattached to each of them. At least one of the types of nanoparticles ofthe aggregate probe has oligonucleotides attached to it which have asequence complementary to a second portion of the sequence of thenucleic acid.

In yet another embodiment, the kit comprises a substrate havingoligonucleotides attached to it. The oligonucleotides have a sequencecomplementary to the sequence of a first portion of a nucleic acid. Thekit further includes a first container holding an aggregate probe. Theaggregate probe comprises at least two types of nanoparticles havingoligonucleotides attached to them. The nanoparticles of the aggregateprobe are bound to each other as a result of the hybridization of someof the oligonucleotides attached to each of them. At least one of thetypes of nanoparticles of the aggregate probe has oligonucleotidesattached thereto which have a sequence complementary to a second portionof the sequence of the nucleic acid.

In an additional embodiment, the kit comprises a substrate havingoligonucleotides attached to it and a first container holding anaggregate probe. The aggregate probe comprises at least two types ofnanoparticles having oligonucleotides attached to them. Thenanoparticles of the aggregate probe are bound to each other as a resultof the hybridization of some of the oligonucleotides attached to each ofthem. At least one of the types of nanoparticles of the aggregate probehas oligonucleotides attached to it which have a sequence complementaryto a first portion of the sequence of the nucleic acid. The kit alsoincludes a second container holding nanoparticles. The nanoparticleshave at least two types of oligonucleotides attached to them. The firsttype of oligonucleotides has a sequence complementary to a secondportion of the sequence of the nucleic acid. The second type ofoligonucleotides has a sequence complementary to at least a portion ofthe sequence of the oligonucleotides attached to the substrate.

In another embodiment, the kit comprises a substrate which hasoligonucleotides attached to it. The oligonucleotides have a sequencecomplementary to the sequence of a first portion of a nucleic acid. Thekit also comprises a first container holding liposomes havingoligonucleotides attached to them. The oligonucleotides have a sequencecomplementary to the sequence of a second portion of the nucleic acid.The kit further includes a second container holding an aggregate probecomprising at least two types of nanoparticles having oligonucleotidesattached to them. The nanoparticles of the aggregate probe are bound toeach other as a result of the hybridization of some of theoligonucleotides attached to each of them. At least one of the types ofnanoparticles of the aggregate probe has oligonucleotides attached to itwhich have a hydrophobic groups attached to the ends not attached to thenanoparticles.

In a further embodiment, the kit may comprise a first container holdingnanoparticles having oligonucleotides attached thereto. The kit alsoincludes one or more additional containers, each container holding abinding oligonucleotide. Each binding oligonucleotide has a firstportion which has a sequence complementary to at least a portion of thesequence of oligonucleotides on the nanoparticles and a second portionwhich has a sequence complementary to the sequence of a portion of anucleic acid to be detected. The sequences of the second portions of thebinding oligonucleotides may be different as long as each sequence iscomplementary to a portion of the sequence of the nucleic acid to bedetected.

In another embodiment, the kit comprises a container holding one type ofnanoparticles having oligonucleotides attached thereto and one or moretypes of binding oligonucleotides. Each of the types of bindingoligonucleotides has a sequence comprising at least two portions. Thefirst portion is complementary to the sequence of the oligonucleotideson the nanoparticles, whereby the binding oligonucleotides arehybridized to the oligonucleotides on the nanoparticles in thecontainer(s). The second portion is complementary to the sequence of aportion of the nucleic acid.

In another embodiment, kits may comprise one or two containers holdingtwo types of particles. The first type of particles havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid. The oligonucleotidesare labeled with an energy donor on the ends not attached to theparticles. The second type of particles having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a secondportion of a nucleic acid. The oligonucleotides are labeled with anenergy acceptor on the ends not attached to the particles. The energydonors and acceptors may be fluorescent molecules.

In a further embodiment, the kit comprises a first container holdingnanoparticles having oligonucleotides attached thereto. The kit alsoincludes one or more additional containers, each container holdingbinding oligonucleotides. Each binding oligonucleotide has a firstportion which has a sequence complementary to at least a portion of thesequence of oligonucleotides on the nanoparticles and a second portionwhich has a sequence complementary to the sequence of a portion of anucleic acid to be detected. The sequences of the second portions of thebinding oligonucleotides may be different as long as each sequence iscomplementary to a portion of the sequence of the nucleic acid to bedetected.

In yet another embodiment, the kit comprises a container holding onetype of nanoparticles having oligonucleotides attached thereto and oneor more types of binding oligonucleotides. Each of the types of bindingoligonucleotides has a sequence comprising at least two portions. Thefirst portion is complementary to the sequence of the oligonucleotideson the nanoparticles, whereby the binding oligonucleotides arehybridized to the oligonucleotides on the nanoparticles in thecontainer(s). The second portion is complementary to the sequence of aportion of the nucleic acid.

In another alternative embodiment, the kit comprises at least threecontainers. The first container holds nanoparticles. The secondcontainer holds a first oligonucleotide having a sequence complementaryto the sequence of a first portion of a nucleic acid. The thirdcontainer holds a second oligonucleotide having a sequence complementaryto the sequence of a second portion of the nucleic acid. The kit mayfurther comprise a fourth container holding a binding oligonucleotidehaving a selected sequence having at least two portions, the firstportion being complementary to at least a portion of the sequence of thesecond oligonucleotide, and a fifth container holding an oligonucleotidehaving a sequence complementary to the sequence of a second portion ofthe binding oligonucleotide.

In another embodiment, the kit comprises one or two containers, thecontainer(s) holding two types of particles. The first type of particleshaving oligonucleotides attached thereto that have a sequencecomplementary to a first portion of the sequence of a nucleic acid andhave energy donor molecules attached to the ends not attached to thenanoparticles. The second type of particles having oligonucleotidesattached thereto that have a sequence complementary to a second portionof the sequence of a nucleic acid and have energy acceptor moleculesattached to the ends not attached to the nanoparticles. The energydonors and acceptors may be fluorescent molecules.

In a further embodiment, the kit comprises a first container holding atype of microspheres having oligonucleotides attached thereto. Theoligonucleotides have a sequence complementary to a first portion of thesequence of a nucleic acid and are labeled with a fluorescent molecule.The kit also comprises a second container holding a type ofnanoparticles having oligonucleotides attached thereto. Theoligonucleotides have a sequence complementary to a second portion ofthe sequence of the nucleic acid.

In another embodiment, the kit comprises a first container holding afirst type of metallic or semiconductor nanoparticles havingoligonucleotides attached thereto. The oligonucleotides have a sequencecomplementary to a first portion of the sequence of a nucleic acid andare labeled with a fluorescent molecule. The kit also comprises a secondcontainer holding a second type of metallic or semiconductornanoparticles having oligonucleotides attached thereto. Theseoligonucleotides have a sequence complementary to a second portion ofthe sequence of a nucleic acid and are labeled with a fluorescentmolecule.

In another embodiment, the kit comprises a container holding anaggregate probe. The aggregate probe comprises at least two types ofnanoparticles having oligonucleotides attached to them. Thenanoparticles of the aggregate probe are bound to each other as a resultof the hybridization of some of the oligonucleotides attached to each ofthem. At least one of the types of nanoparticles of the aggregate probehas oligonucleotides attached to it which have a sequence complementaryto a portion of the sequence of a nucleic acid.

In an additional embodiment, the kit comprises a container holding anaggregate probe. The aggregate probe comprises at least two types ofnanoparticles having oligonucleotides attached to them. Thenanoparticles of the aggregate probe are bound to each other as a resultof the hybridization of some of the oligonucleotides attached to each ofthem. At least one of the types of nanoparticles of the aggregate probehas oligonucleotides attached to it which have a hydrophobic groupattached to the end not attached to the nanoparticles.

In a further embodiment, the kit comprises a container holding asatellite probe. The satellite probe comprises a particle havingattached thereto oligonucleotides. The oligonucleotides have a firstportion and a second portion, both portions having sequencescomplementary to portions of the sequence of a nucleic acid. Thesatellite probe also comprises probe oligonucleotides hybridized to theoligonucleotides attached to the nanoparticles. The probeoligonucleotides have a first portion and a second portion. The firstportion has a sequence complementary to the sequence of the firstportion of the oligonucleotides attached to the particles, and bothportions have sequences complementary to portions of the sequence of thenucleic acid. The probe oligonucleotides also have a reporter moleculeattached to one end.

In another embodiment, the kit comprising a container holding a coreprobe, the core probe comprising at least two types of nanoparticleshaving oligonucleotides attached thereto, the nanoparticles of the coreprobe being bound to each other as a result of the hybridization of someof the oligonucleotides attached to them.

In yet another embodiment, the kit comprises a substrate having attachedto it at least one pair of electrodes with oligonucleotides attached tothe substrate between the electrodes. The oligonucleotides have asequence complementary to a first portion of the sequence of a nucleicacid to be detected.

The invention also provides the satellite probe, an aggregate probe anda core probe.

The invention further provides a substrate having nanoparticles attachedthereto. The nanoparticles may have oligonucleotides attached theretowhich have a sequence complementary to the sequence of a first portionof a nucleic acid.

The invention also provides a metallic or semiconductor nanoparticlehaving oligonucleotides attached thereto. The oligonucleotides arelabeled with fluorescent molecules at the ends not attached to thenanoparticle.

The invention further provides a method of nanofabrication. The methodcomprises providing at least one type of linking oligonucleotide havinga selected sequence, the sequence of each type of linkingoligonucleotide having at least two portions. The method furthercomprises providing one or more types of nanoparticles havingoligonucleotides attached thereto, the oligonucleotides on each type ofnanoparticles having a sequence complementary to a portion of thesequence of a linking oligonucleotide. The linking oligonucleotides andnanoparticles are contacted under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles to thelinking oligonucleotides so that a desired nanomaterials ornanostructure is formed.

The invention provides another method of nanofabrication. This methodcomprises providing at least two types of nanoparticles havingoligonucleotides attached thereto. The oligonucleotides on the firsttype of nanoparticles have a sequence complementary to that of theoligonucleotides on the second type of nanoparticles. Theoligonucleotides on the second type of nanoparticles have a sequencecomplementary to that of the oligonucleotides on the first type ofnanoparticle-oligonucleotide conjugates. The first and second types ofnanoparticles are contacted under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles to each otherso that a desired nanomaterials or nanostructure is formed.

The invention further provides nanomaterials or nanostructures composedof nanoparticles having oligonucleotides attached thereto, thenanoparticles being held together by oligonucleotide connectors.

The invention also provides a composition comprising at least two typesof nanoparticles having oligonucleotides attached thereto. Theoligonucleotides on the first type of nanoparticles have a sequencecomplementary to the sequence of a first portion of a nucleic acid or alinking oligonucleotide. The oligonucleotides on the second type ofnanoparticles have a sequence complementary to the sequence of a secondportion of the nucleic acid or linking oligonucleotide.

The invention further provides an assembly of containers comprising afirst container holding nanoparticles having oligonucleotides attachedthereto, and a second container holding nanoparticles havingoligonucleotides attached thereto. The oligonucleotides attached to thenanoparticles in the first container have a sequence complementary tothat of the oligonucleotides attached to the nanoparticles in the secondcontainer. The oligonucleotides attached to the nanoparticles in thesecond container have a sequence complementary to that of theoligonucleotides attached to the nanoparticles in the first container.

The invention also provides a nanoparticle having a plurality ofdifferent oligonucleotides attached to it.

The invention further provides a method of separating a selected nucleicacid having at least two portions from other nucleic acids. The methodcomprises providing one or more types of nanoparticles havingoligonucleotides attached thereto, the oligonucleotides on each of thetypes of nanoparticles having a sequence complementary to the sequenceof one of the portions of the selected nucleic acid. The selectednucleic acid and other nucleic acids are contacted with thenanoparticles under conditions effective to allow hybridization of theoligonucleotides on the nanoparticles with the selected nucleic acid sothat the nanoparticles hybridized to the selected nucleic acid aggregateand precipitate.

In addition, the invention provides methods of making uniquenanoparticle-oligonucleotide conjugates. The first such method comprisesbinding oligonucleotides to charged nanoparticles to produce stablenanoparticle-oligonucleotide conjugates. To do so, oligonucleotideshaving covalently bound thereto a moiety comprising a functional groupwhich can bind to the nanoparticles are contacted with the nanoparticlesin water for a time sufficient to allow at least some of theoligonucleotides to bind to the nanoparticles by means of the functionalgroups. Next, at least one salt is added to the water to form a saltsolution. The ionic strength of the salt solution must be sufficient toovercome at least partially the electrostatic repulsion of theoligonucleotides from each other and, either the electrostaticattraction of the negatively-charged oligonucleotides forpositively-charged nanoparticles, or the electrostatic repulsion of thenegatively-charged oligonucleotides from negatively-chargednanoparticles. After adding the salt, the oligonucleotides andnanoparticles are incubated in the salt solution for an additionalperiod of time sufficient to allow sufficient additionaloligonucleotides to bind to the nanoparticles to produce the stablenanoparticle-oligonucleotide conjugates. The invention also includes thestable nanoparticle-oligonucleotide conjugates, methods of using theconjugates to detect and separate nucleic acids, kits comprising theconjugates, methods of nanofabrication using the conjugates, andnanomaterials and nanostructures comprising the conjugates.

The invention provides another method of binding oligonucleotides tonanoparticles to produce nanoparticle-oligonucleotide conjugates. Themethod comprises providing oligonucleotides, the oligonucleotidescomprising a type of recognition oligonucleotides and a type of diluentoligonucleotides. The oligonucleotides and the nanoparticles arecontacted under conditions effective to allow at least some of each ofthe types of oligonucleotides to bind to the nanoparticles to producethe conjugates. The invention also includes thenanoparticle-oligonucleotide conjugates produced by this method, methodsof using the conjugates to detect and separate nucleic acids, kitscomprising the conjugates, methods of nanofabrication using theconjugates, and nanomaterials and nanostructures comprising theconjugates. “Recognition oligonucleotides” are oligonucleotides whichcomprise a sequence complementary to at least a portion of the sequenceof a nucleic acid or oligonucleotide target. “Diluent oligonucleotides”may have any sequence which does not interfere with the ability of therecognition oligonucleotides to be bound to the nanoparticles or to bindto their targets.

The invention provides yet another method of binding oligonucleotides tonanoparticles to produce nanoparticle-oligonucleotide conjugates. Themethod comprises providing oligonucleotides, the oligonucleotidescomprising at least one type of recognition oligonucleotides. Therecognition oligonucleotides comprise a recognition portion and a spacerportion. The recognition portion of the recognition oligonucleotides hasa sequence complementary to at least one portion of the sequence of anucleic acid or oligonucleotide target. The spacer portion of therecognition oligonucleotide is designed so that it can bind to thenanoparticles. As a result of the binding of the spacer portion of therecognition oligonucleotide to the nanoparticles, the recognitionportion is spaced away from the surface of the nanoparticles and is moreaccessible for hybridization with its target. To make the conjugates,the oligonucleotides, including the recognition oligonucleotides, andthe nanoparticles are contacted under conditions effective allow atleast some of the recognition oligonucleotides to bind to thenanoparticles. The invention also includes thenanoparticle-oligonucleotide conjugates produced by this method, methodsof using the conjugates to detect and separate nucleic acids, kitscomprising the conjugates, methods of nanofabrication using theconjugates, and nanomaterials and nanostructures comprising theconjugates.

As used herein, a “type of oligonucleotides” refers to a plurality ofoligonucleotide molecules having the same sequence. A “type of”nanoparticles, conjugates, particles, latex microspheres, etc. havingoligonucleotides attached thereto refers to a plurality of that itemhaving the same type(s) of oligonucleotides attached to them.“Nanoparticles having oligonucleotides attached thereto” are alsosometimes referred to as “nanoparticle-oligonucleotide conjugates” or,in the case of the detection methods of the invention,“nanoparticle-oligonucleotide probes,” “nanoparticle probes,” or just“probes.”

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic diagram illustrating the formation of nanoparticleaggregates by combining nanoparticles having complementaryoligonucleotides attached to them, the nanoparticles being held togetherin the aggregates as a result of the hybridization of the complementaryoligonucleotides. X represents any covalent anchor (such as—S(CH₂)₃OP(O)(O⁻)—, where S is joined to a gold nanoparticle). For thesake of simplicity in FIG. 1 and some subsequent figures, only oneoligonucleotide is shown to be attached to each particle but, in fact,each particle has several oligonucleotides attached to it. Also, it isimportant to note that in FIG. 1 and subsequent figures, the relativesizes of the gold nanoparticles and the oligonucleotides are not drawnto scale.

FIG. 2: Schematic diagram illustrating a system for detecting nucleicacid using nanoparticles having oligonucleotides attached thereto. Theoligonucleotides on the two nanoparticles have sequences complementaryto two different portions of the single-stranded DNA shown. As aconsequence, they hybridize to the DNA producing detectable changes(forming aggregates and producing a color change).

FIG. 3: Schematic diagram of a variation of the system shown in FIG. 2.The oligonucleotides on the two nanoparticles have sequencescomplementary to two different portions of the single-stranded DNA shownwhich are separated by a third portion which is not complementary to theoligonucleotides on the nanoparticles. Also shown is an optional filleroligonucleotide which can be used to hybridize with the noncomplementaryportion of the single-stranded DNA. When the DNA, nanoparticles andfiller oligonucleotides are combined, the nanoparticles aggregate, withthe formation of nicked, double-stranded oligonucleotide connectors.

FIG. 4: Schematic diagram illustrating reversible aggregation ofnanoparticles having oligonucleotides attached thereto as a result ofhybridization and de-hybridization with a linking oligonucleotide. Theillustrated linking oligonucleotide is a double-stranded DNA havingoverhanging termini (sticky ends) which are complementary to theoligonucleotides attached to the nanoparticles.

FIG. 5: Schematic diagram illustrating the formation of nanoparticleaggregates by combining nanoparticles having oligonucleotides attachedthereto with linking oligonucleotides having sequences complementary tothe oligonucleotides attached to the nanoparticles.

FIG. 6: Cuvettes containing two types of gold colloids, each having adifferent oligonucleotide attached thereto and a linking double-strandedoligonucleotide with sticky ends complementary to the oligonucleotidesattached to the nanoparticles (see FIG. 4). Cuvette A—at 80° C., whichis above the Tm of the linking DNA; de-hybridized (thermally denatured).The color is dark red. Cuvette B—after cooling to room temperature,which is below the Tm of the linking DNA; hybridization has taken place,and the nanoparticles have aggregated, but the aggregates have notprecipitated. The color is purple. Cuvette C—after several hours at roomtemperature, the aggregated nanoparticles have settled to the bottom ofthe cuvette. The solution is clear, and the precipitate is pinkish gray.Heating B or C will result in A.

FIG. 7: A graph of absorbance versus wavelength in nm showing changes inabsorbance when gold nanoparticles having oligonucleotides attachedthereto aggregate due to hybridization with linking oligonucleotidesupon lowering of the temperature, as illustrated in FIG. 4.

FIGS. 8A–B: FIG. 8A is a graph of change in absorbance versustemperature/time for the system illustrated in FIG. 4. At lowtemperatures, gold nanoparticles having oligonucleotides attachedthereto aggregate due to hybridization with linking oligonucleotides(see FIG. 4). At high temperature (80° C.), the nanoparticles arede-hybridized. Changing the temperature over time shows that this is areversible process. FIG. 8B is a graph of change in absorbance versustemperature/time performed in the same manner using an aqueous solutionof unmodified gold nanoparticles. The reversible changes seen in FIG. 8Aare not observed.

FIGS. 9A–B: Transmission Electron Microscope (TEM) images. FIG. 9A is aTEM image of aggregated gold nanoparticles held together byhybridization of the oligonucleotides on the gold nanoparticles withlinking oligonucleotides. FIG. 9B is a TEM image of a two-dimensionalaggregate showing the ordering of the linked nanoparticles.

FIG. 10: Schematic diagram illustrating the formation ofthermally-stable triple-stranded oligonucleotide connectors betweennanoparticles having the pyrimidine:purine:pyrimidine motif. Suchtriple-stranded connectors are stiffer than double-stranded connectors.In FIG. 10, one nanoparticle has an oligonucleotide attached to it whichis composed of all purines, and the other nanoparticle has anoligonucleotide attached to it which is composed of all pyrimidines. Thethird oligonucleotide for forming the triple-stranded connector (notattached to a nanoparticle) is composed of pyrimidines.

FIG. 11: Schematic diagram illustrating the formation of nanoparticleaggregates by combining nanoparticles having complementaryoligonucleotides attached to them, the nanoparticles being held togetherin the aggregates as a result of the hybridization of the complementaryoligonucleotides. In FIG. 11, the circles represent the nanoparticles,the formulas are oligonucleotide sequences, and s is the thio-alkyllinker. The multiple oligonucleotides on the two types of nanoparticlescan hybridize to each other, leading to the formation of an aggregatestructure.

FIGS. 12A–F: Schematic diagrams illustrating systems for detectingnucleic acid using nanoparticles having oligonucleotides attachedthereto. Oligonucleotide-nanoparticle conjugates 1 and 2 andsingle-stranded oligonucleotide targets 3, 4, 5, 6 and 7 areillustrated. The circles represent the nanoparticles, the formulas areoligonucleotide sequences, and the dotted and dashed lines representconnecting links of nucleotide.

FIGS. 13A–B: Schematic diagrams illustrating systems for detecting DNA(analyte DNA) using nanoparticles and a transparent substrate.

FIGS. 14A–B: FIG. 14A is a graph of absorbance versus wavelength in nmshowing changes in absorbance when gold nanoparticles havingoligonucleotides attached thereto (one population of which is insolution and one population of which is attached to a transparentsubstrate as illustrated in FIG. 13B) aggregate due to hybridizationwith linking oligonucleotides. FIG. 14B a graph of change in absorbancefor the hybridized system referred to in FIG. 14A as the temperature isincreased (melted).

FIGS. 15A–G: Schematic diagrams illustrating systems for detectingnucleic acid using nanoparticles having oligonucleotides attachedthereto. Oligonucleotide-nanoparticle conjugates 1 and 2 andsingle-stranded oligonucleotide targets 3, 4, 5, 6, 7 and 8 areillustrated. The circles represent the nanoparticles, the formulas areoligonucleotide sequences, and S represents the thio-alkyl linker.

FIGS. 16A–C: Schematic diagrams illustrating systems for detectingnucleic acid using nanoparticles having oligonucleotides attachedthereto. Oligonucleotide-nanoparticle conjugates 1 and 2,single-stranded oligonucleotide targets of different lengths, and filleroligonucleotides of different lengths are illustrated. The circlesrepresent the nanoparticles, the formulas are oligonucleotide sequences,and S represents the thio-alkyl linker.

FIGS. 17A–E: Schematic diagrams illustratingnanoparticle-oligonucleotide conjugates and systems for detectingnucleic acid using nanoparticles having oligonucleotides attachedthereto. The circles represent the nanoparticles, the straight linesrepresent oligonucleotide chains (bases not shown), two closely-spacedparallel lines represent duplex segments, and the small letters indicatespecific nucleotide sequences (a is complementary to a′, b iscomplementary to b′, etc.).

FIG. 18: Schematic diagram illustrating a system for detecting nucleicacid using liposomes (large double circle), nanoparticles (small opencircles) and a transparent substrate. The filled-in squares representcholesteryl groups, the squiggles represent oligonucleotides, and theladders represent double-stranded (hybridized) oligonucleotides.

FIGS. 19A–B: FIG. 19A is a graph of absorbance versus wavelength in nmshowing changes in absorbance when gold nanoparticle-oligonucleotideconjugates assemble in multiple layers on a transparent substrate asillustrated in FIG. 13A. FIG. 19B is a graph of change in absorbance forthe hybridized system referred to in FIG. 19A as the temperature isincreased (melted).

FIGS. 20A–B: Illustrations of schemes using fluorescent-labeledoligonucleotides attached to metallic or semiconductor quenchingnanoparticles (FIG. 20A) or to non-metallic, non-semiconductor particles(FIG. 20B).

FIG. 21: Schematic diagram illustrating a system for detecting targetnucleic acid using gold nanoparticles having oligonucleotides attachedthereto and latex microspheres having fluorescently-labeledoligonucleotides attached thereto. The small, closed, dark circlesrepresent the nanoparticles, the large, open circles represent the latexmicrospheres, and the large oval represents a microporous membrane.

FIG. 22: Schematic diagram illustrating a system for detecting targetnucleic acid using two types of fluorescently-labeledoligonucleotide-nanoparticle conjugates. The closed circles representthe nanoparticles, and the large oval represents a microporous membrane.

FIG. 23: Sequences of materials utilized in an assay for AnthraxProtective Antigen (see Example 12).

FIG. 24: Schematic diagram illustrating a system for detecting targetnucleic acid using a “satellite probe” which comprises magneticnanoparticles (dark spheres) having oligonucleotides (straight lines)attached to them, probe oligonucleotides (straight lines) hybridized tothe oligonucleotides attached to the nanoparticles, the probeoligonucleotides being labeled with a reporter group (open rectangularbox). A, B, C, A′, B′, and C′ represent specific nucleotide sequences,with A, B and C being complementary to A′, B′ and C′, respectively.

FIGS. 25A–B: Schematic diagrams illustrating systems for detecting DNAusing nanoparticles and a transparent substrate. In these figures, a, band c refer to different oligonucleotide sequences, and a′, b′ and c′refer to oligonucleotide sequences complementary to a, b and c,respectively.

FIG. 26: Schematic diagram illustrating systems for forming assembliesof CdSe/ZnS core/shell quantum dots (QD).

FIGS. 27A–D: FIG. 27A shows fluorescence spectra comparing dispersed andaggregated QDs, with an excitation at 400 nm. The samples were preparedidentically, except for the addition of complementary “linker” DNA toone and an equal volume and concentration of non-complementary DNA tothe other. FIG. 27B shows UV-Visible spectra of QD/QD assemblies atdifferent temperatures before, during and after “melting”. FIG. 27Cshows high resolution TEM image of a portion of a hybrid gold/QDassembly. The lattice fringes of the QDs, which resemble fingerprints,appear near each gold nanoparticle. FIG. 27D shows UV-Visible spectra ofhybrid gold/QD assemblies at different temperatures before, during andafter “melting”. The insets in FIGS. 27B and 27D display temperatureversus extinction profiles for the thermal denaturation of theassemblies. Denturation experiments were conducted in 0.3 M NaCl, 10 mMphosphate buffer (pH 7), 0.01% sodium azide with 13 nm goldnanoparticles and/or ˜4 nm CdSe/ZnS core/shell QDs.

FIGS. 28A–E: Schematic diagrams illustrating the preparation of coreprobes, aggregate probes and systems for detecting DNA using theseprobes. In these figures, a, b, c and d refer to differentoligonucleotide sequences, and a′, b′, c′ and d′ refer tooligonucleotide sequences complementary to a, b, c and d, respectively.

FIG. 29: Graph of fractional displacement of oligonucleotides bymercaptoethanol from nanoparticles (closed circles) or gold thin films(open squares) to which the oligonucleotides had been attached.

FIG. 30: Graph of surface coverages of recognition oligonucleotides onnanoparticles obtained for different ratios of recognition:diluentoligonucleotides used in the preparation of thenanoparticle-oligonucleotide conjugates.

FIG. 31: Graph of surface coverages of hybridized complementaryoligonucleotides versus different surface coverages of recognitionoligonucleotides on nanoparticles.

FIG. 32: Schematic diagram illustrating system for detecting a targetDNA in a four-element array on a substrate usingnanoparticle-oligonucleotide conjugates and amplification with silverstaining.

FIG. 33: Images obtained with a flatbed scanner of 7 mm×13 mmoligonucleotide-functionalized float glass slides. (A) Slide beforehybridization of DNA target and gold nanoparticle-oligonucleotideindicator conjugate. (B) Slide A after hybridization of 10 nM target DNAand 5 nM nanoparticle-oligonucleotide indicator conjugate. A pink colorwas imparted by attached, red 13 nm diameter gold nanoparticles. (C)Slide B after exposure to silver amplification solution for 5 minutes.(D) Same as (A). (E) Slide D after hybridization of 100 pM target and 5nM nanoparticle-oligonucleotide indicator conjugate. The absorbance ofthe nanoparticle layer was too low to be observed with the naked eye orflatbed scanner. (F) Slide E after exposure to silver amplificationsolution for 5 minutes. Note that slide F is much lighter than slide C,indicating lower target concentration. (G) Control slide, exposed to 5nM nanoparticle-oligonucleotide indicator conjugate and exposed tosilver amplification solution for 5 minutes. No darkening of the slidewas observed.

FIG. 33: Graph of greyscale (optical density) ofoligonucleotide-functionalized glass surface exposed to varyingconcentrations of target DNA, followed by 5 nM gold ofnanoparticle-oligonucleotide indicator conjugates and silveramplification for 5 minutes.

FIGS. 35A–B: Graphs of percent hybridized label versus temperatureshowing dissociation of fluorophore-labeled (FIG. 35A) andnanoparticle-labeled (FIG. 35B) targets from anoligonucleotide-functionalized glass surface. Measurements were made bymeasuring fluorescence (FIG. 35A) or absorbance (FIG. 35B) ofdissociated label in the solution above the glass surface. The lineslabeled “b” show the dissociation curves for perfectly matchedoligonucleotides on the glass, and the lines labeled “r” show curves formismatched oligonucleotides (a one-base mismatch) on the glass. Verticallines in the graphs illustrate the fraction of target dissociated at agiven temperature (halfway between the melting temperatures T_(m) ofeach curve) for each measurement, and the expected selectivity ofsequence identification for fluorophore- and nanoparticle-based genechips. Fluorescence (FIG. 35A): complement (69%)/mismatch (38%)=1.8:1.Absorbance (FIG. 35B): complement (85%)/mismatch (14%)=6:1. The breadthof the fluorophore-labeled curves (FIG. 35A) is characteristic of thedissociation of fluorophore-labeled targets from gene chips (Forman etal., in Molecular Modeling of Nucleic Acids, Leontis et al., eds., (ACSSymposium Series 682, American Chemical Society, Washington D.C., 1998),pages 206–228).

FIGS. 36A–B: Images of model oligonucleotide arrays challenged withsynthetic target and fluorescent-labeled (FIG. 36A) ornanoparticle-labeled (FIG. 36B) nanoparticle-oligonucleotide conjugateprobes. C, A, T, and G represent spots (elements) on the array where asingle base change has been made in the oligonucleotide attached to thesubstrate to give a perfect match with the target (base A) or a singlebase mismatch (base C, T or G in place of the perfect match with baseA). The greyscale ratio for elements C:A:T:G is 9:37:9:11 for FIGS. 36Aand 3:62:7:34 for FIG. 36B.

FIG. 37: Schematic diagram illustrating system for forming aggregates(A) or layers (B) of nanoparticles (a and b) linked by a linking nucleicacid (3).

FIG. 38A: UV-visible spectra of alternating layers of gold nanoparticlesa and b (see FIG. 37) hybridized to an oligonucleotide-functionalizedglass microscope slide via the complementary linker 3. The spectra arefor assemblies with 1 (a, λ_(max)=524 nm), 2 (b, λ_(max)=529 nm), 3 (c,λ_(max)=532 nm), 4 (d, λ_(max)=534 nm) or 5 (e, λ_(max)=534 nm) layers.These spectra were measured directly through the slide.

FIG. 38B: Graph of absorbance for nanoparticle assemblies (see FIG. 38A)at λ_(max) with increasing numbers of layers.

FIGS. 39A–F: FIG. 39A: FE-SEM of one layer ofoligonucleotide-functionalized gold nanoparticles cohybridized with DNAlinker to an oligonucleotide-functionalized, conductive indium-tin-oxide(ITO) slide (prepared in the same way as oligonucleotide-funcationalizedglass slide). The visible absorbance spectrum of this slide wasidentical to FIG. 38A, indicating that functionalization andnanoparticle coverage on ITO is similar to that on glass. The averagedensity of counted nanoparticles from 10 such images was approximately800 nanoparticles/μm². FIG. 39B: FE-SEM image of two layers ofnanoparticles on the ITO slide. The average density of countednanoparticles from 10 such images was approximately 2800 particles/μm².FIG. 39C: Absorbance at 260 nm (A₂₆₀) showing dissociation of a 0.5 μMsolution of the oligonucleotide duplex (1+2+3; see FIG. 37, A) to singlestrands in 0.3 M NaCl, 10 mM phosphate buffer solution (pH 7). FIGS.39D–F: Absorbance at 260 nm (A₂₆₀) showing dissociation of 1 layer (FIG.39D), 4 layers (FIG. 39E) and 10 layers (FIG. 39F) ofoligonucleotide-functionalized gold nanoparticles from glass slidesimmersed in 0.3 M NaCl, 10 mM phosphate buffer solution. Meltingprofiles were obtained by measuring the decreasing absorption at 520 nm(A₅₂₀) through the slides with increasing temperature. In each of FIGS.39D–F, the insets show the first derivatives of the measureddissociation curves. FWHM of these curves were (FIG. 39C inset) 13.2°C., (FIG. 39D inset) 5.6° C., (FIG. 39E inset) 3.2° C., and (FIG. 39Finset) 2.9° C.

FIG. 40: Schematic diagram illustrating system used to measure theelectrical properties of gold nanoparticle assemblies linked by DNA. Forsimplicity, only one hybridization event is drawn.

FIG. 41: Schematic diagram illustrating a method of detecting nucleicacid using gold electrodes and gold nanoparticles.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

Nanoparticles useful in the practice of the invention include metal(e.g., gold, silver, copper and platinum), semiconductor (e.g., CdSe,CdS, and CdS or CdSe coated with ZnS) and magnetic (e.g.,ferromagnetite) colloidal materials. Other nanoparticles useful in thepractice of the invention include ZnS, ZnO, TiO₂, AgI, AgBr, HgI₂, PbS,PbSe, ZnTe, CdTe, In₂S₃, In₂Se₃, Cd₃P₂, Cd₃As₂, InAs, and GaAs. The sizeof the nanoparticles is preferably from about 5 nm to about 150 nm (meandiameter), more preferably from about 5 to about 50 nm, most preferablyfrom about 10 to about 30 nm. The nanoparticles may also be rods.

Methods of making metal, semiconductor and magnetic nanoparticles arewell-known in the art. See, e.g., Schmid, G. (ed.) Clusters and Colloids(VCH, Weinheim, 1994); Hayat, M. A. (ed.) Colloidal Gold: Principles,Methods, and Applications (Academic Press, San Diego, 1991); Massart,R., IEEE Taransactions On Magnetics, 17, 1247 (1981); Ahmadi, T. S. etal., Science, 272, 1924 (1996); Henglein, A. et al., J. Phys. Chem., 99,14129 (1995); Curtis, A. C., et al., Angew. Chem. Int. Ed. Engl., 27,1530 (1988).

Methods of making ZnS, ZnO, TiO₂, AgI, AgBr, HgI₂, PbS, PbSe, ZnTe,CdTe, In₂S₃, In₂Se₃, Cd₃P₂, Cd₃As₂, InAs, and GaAs nanoparticles arealso known in the art. See, e.g., Weller, Angew. Chem. Int. Ed. Engl.,32, 41 (1993); Henglein, Top. Curr. Chem., 143, 113 (1988); Henglein,Chem. Rev., 89, 1861 (1989); Brus, Appl. Phys. A., 53, 465 (1991);Bahncmann, in Photochemical Conversion and Storage of Solar Energy (eds.Pelizetti and Schiavello 1991), page 251; Wang and Herron, J. Phys.Chem., 95, 525 (1991); Olshavsky et al., J. Am. Chem. Soc., 112, 9438(1990); Ushida et al., J. Phys. Chem., 95, 5382 (1992).

Suitable nanoparticles are also commercially available from, e.g., TedPella, Inc. (gold), Amersham Corporation (gold) and Nanoprobes, Inc.(gold).

Presently preferred for use in detecting nucleic acids are goldnanoparticles. Gold colloidal particles have high extinctioncoefficients for the bands that give rise to their beautiful colors.These intense colors change with particle size, concentration,interparticle distance, and extent of aggregation and shape (geometry)of the aggregates, making these materials particularly attractive forcalorimetric assays. For instance, hybridization of oligonucleotidesattached to gold nanoparticles with oligonucleotides and nucleic acidsresults in an immediate color change visible to the naked eye (see,e.g., the Examples).

Gold nanoparticles are also presently preferred for use innanofabrication for the same reasons given above and because of theirstability, ease of imaging by electron microscopy, andwell-characterized modification with thiol functionalities (see below).Also preferred for use in nanofabrication are semiconductornanoparticles because of their unique electronic and luminescentproperties.

The nanoparticles, the oligonucleotides or both are functionalized inorder to attach the oligonucleotides to the nanoparticles. Such methodsare known in the art. For instance, oligonucleotides functionalized withalkanethiols at their 3′-termini or 5′-termini readily attach to goldnanoparticles. See Whitesides, Proceedings of the Robert A. WelchFoundation 39th Conference On Chemical Research Nanophase Chemistry,Houston, Tex., pages 109–121 (1995). See also, Mucic et al. Chem.Commun. 555–557 (1996) (describes a method of attaching 3′ thiol DNA toflat gold surfaces; this method can be used to attach oligonucleotidesto nanoparticles). The alkanethiol method can also be used to attacholigonucleotides to other metal, semiconductor and magnetic colloids andto the other nanoparticles listed above. Other functional groups forattaching oligonucleotides to solid surfaces include phosphorothioategroups (see, e.g., U.S. Pat. No. 5,472,881 for the binding ofoligonucleotide-phosphorothioates to gold surfaces), substitutedalkylsiloxanes (see, e.g. Burwell, Chemical Technology, 4, 370–377(1974) and Matteucci and Caruthers, J. Am. Chem. Soc., 103, 3185–3191(1981) for binding of oligonucleotides to silica and glass surfaces, andGrabar et al., Anal. Chem., 67, 735–743 for binding ofaminoalkylsiloxanes and for similar binding of mercaptoaklylsiloxanes).Oligonucleotides terminated with a 5′ thionucleoside or a 3′thionucleoside may also be used for attaching oligonucleotides to solidsurfaces. The following references describe other methods which may beemployed to attached oligonucleotides to nanoparticles: Nuzzo et al., J.Am. Chem. Soc., 109, 2358 (1987) (disulfides on gold); Allara and Nuzzo,Langmuir, 1, 45 (1985) (carboxylic acids on aluminum); Allara andTompkins, J. Colloid Interface Sci., 49,410–421 (1974) (carboxylic acidson copper); Iler, The Chemistry Of Silica, Chapter 6, (Wiley 1979)(carboxylic acids on silica); Timmons and Zisman, J. Phys. Chem., 69,984–990 (1965) (carboxylic acids on platinum); Soriaga and Hubbard, J.Am. Chem. Soc., 104, 3937 (1982) (aromatic ring compounds on platinum);Hubbard, Acc. Chem. Res., 13, 177 (1980) (sulfolanes, sulfoxides andother functionalized solvents on platinum); Hickman et al., J. Am. Chem.Soc., 111, 7271 (1989) (isonitriles on platinum); Maoz and Sagiv,Langmuir, 3, 1045 (1987) (silanes on silica); Maoz and Sagiv, Langmuir,3, 1034 (1987) (silanes on silica); Wasserman et al., Langmuir, 5, 1074(1989) (silanes on silica); Eltekova and Eltekov, Langmuir, 3, 951(1987) (aromatic carboxylic acids, aldehydes, alcohols and methoxygroups on titanium dioxide and silica); Lec et al., J. Phys. Chem., 92,2597 (1988) (rigid phosphates on metals).

Each nanoparticle will have a plurality of oligonucleotides attached toit. As a result, each nanoparticle-oligonucleotide conjugate can bind toa plurality of oligonucleotides or nucleic acids having thecomplementary sequence.

Oligonucleotides of defined sequences are used for a variety of purposesin the practice of the invention. Methods of making oligonucleotides ofa predetermined sequence are well-known. See, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein(ed.) Oligonucleotides and Analogues, 1st Ed. (Oxford University Press,New York, 1991). Solid-phase synthesis methods are preferred for botholigoribonucleotides and oligodeoxyribonucleotides (the well-knownmethods of synthesizing DNA are also useful for synthesizing RNA).Oligoribonucleotides and oligodeoxyribonucleotides can also be preparedenzymatically.

The invention provides methods of detecting nucleic acids. Any type ofnucleic acid may be detected, and the methods may be used, e.g., for thediagnosis of disease and in sequencing of nucleic acids. Examples ofnucleic acids that can be detected by the methods of the inventioninclude genes (e.g., a gene associated with a particular disease), viralRNA and DNA, bacterial DNA, fungal DNA, cDNA, mRNA, RNA and DNAfragments, oligonucleotides, synthetic oligonucleotides, modifiedoligonucleotides, single-stranded and double-stranded nucleic acids,natural and synthetic nucleic acids, etc. Thus, examples of the uses ofthe methods of detecting nucleic acids include: the diagnosis and/ormonitoring of viral diseases (e.g., human immunodeficiency virus,hepatitis viruses, herpes viruses, cytomegalovirus, and Epstein-Barrvirus), bacterial diseases (e.g., tuberculosis, Lyme disease, H. pylori,Escherichia coli infections, Legionella infections, Mycoplasmainfections, Salmonella infections), sexually transmitted diseases (e.g.,gonorrhea), inherited disorders (e.g., cystic fibrosis, Duchene musculardystrophy, phenylketonuria, sickle cell anemia), and cancers (e.g.,genes associated with the development of cancer); in forensics; in DNAsequencing; for paternity testing; for cell line authentication; formonitoring gene therapy; and for many other purposes.

The methods of detecting nucleic acids based on observing a color changewith the naked eye are cheap, fast, simple, robust (the reagents arestable), do not require specialized or expensive equipment, and littleor no instrumentation is required. This makes them particularly suitablefor use in, e.g., research and analytical laboratories in DNAsequencing, in the field to detect the presence of specific pathogens,in the doctor's office for quick identification of an infection toassist in prescribing a drug for treatment, and in homes and healthcenters for inexpensive first-line screening.

The nucleic acid to be detected may be isolated by known methods, or maybe detected directly in cells, tissue samples, biological fluids (e.g.,saliva, urine, blood, serum), solutions containing PCR components,solutions containing large excesses of oligonucleotides or highmolecular weight DNA, and other samples, as also known in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.1989) and B. D. Hames and S. J. Higgins, Eds., Gene Probes 1 (IRL Press,New York, 1995). Methods of preparing nucleic acids for detection withhybridizing probes are well known in the art. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and B. D.Hames and S. J. Higgins, Eds., Gene Probes 1 (IRL Press, New York,1995).

If a nucleic acid is present in small amounts, it may be applied bymethods known in the art. See, e.g., Sambrook et al., Molecular Cloning:A Laboratory Manual (2nd ed. 1989) and B. D. Hames and S. J. Higgins,Eds., Gene Probes 1 (IRL Press, New York, 1995). Preferred is polymerasechain reaction (PCR) amplification.

One method according to the invention for detecting nucleic acidcomprises contacting a nucleic acid with one or more types ofnanoparticles having oligonucleotides attached thereto. The nucleic acidto be detected has at least two portions. The lengths of these portionsand the distance(s), if any, between them are chosen so that when theoligonucleotides on the nanoparticles hybridize to the nucleic acid, adetectable change occurs. These lengths and distances can be determinedempirically and will depend on the type of particle used and its sizeand the type of electrolyte which will be present in solutions used inthe assay (as is known in the art, certain electrolytes affect theconformation of nucleic acids).

Also, when a nucleic acid is to be detected in the presence of othernucleic acids, the portions of the nucleic acid to which theoligonucleotides on the nanoparticles are to bind must be chosen so thatthey contain sufficient unique sequence so that detection of the nucleicacid will be specific. Guidelines for doing so are well known in theart.

Although nucleic acids may contain repeating sequences close enough toeach other so that only one type of oligonucleotide-nanoparticleconjugate need be used, this will be a rare occurrence. In general, thechosen portions of the nucleic acid will have different sequences andwill be contacted with nanoparticles carrying two or more differentoligonucleotides, preferably attached to different nanoparticles. Anexample of a system for the detection of nucleic acid is illustrated inFIG. 2. As can be seen, a first oligonucleotide attached to a firstnanoparticle has a sequence complementary to a first portion of thetarget sequence in the single-stranded DNA. A second oligonucleotideattached to a second nanoparticle has a sequence complementary to asecond portion of the target sequence in the DNA. Additional portions ofthe DNA could be targeted with corresponding nanoparticles. See FIG. 17.Targeting several portions of a nucleic acid increases the magnitude ofthe detectable change.

The contacting of the nanoparticle-oligonucleotide conjugates with thenucleic acid takes place under conditions effective for hybridization ofthe oligonucleotides on the nanoparticles with the target sequence(s) ofthe nucleic acid. These hybridization conditions are well known in theart and can readily be optimized for the particular system employed.See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nded. 1989). Preferably stringent hybridization conditions are employed.

Faster hybridization can be obtained by freezing and thawing a solutioncontaining the nucleic acid to be detected and thenanoparticle-oligonucleotide conjugates. The solution may be frozen inany convenient manner, such as placing it in a dry ice-alcohol bath fora sufficient time for the solution to freeze (generally about 1 minutefor 100 μL of solution). The solution must be thawed at a temperaturebelow the thermal denaturation temperature, which can conveniently beroom temperature for most combinations of nanoparticle-oligonucleotideconjugates and nucleic acids. The hybridization is complete, and thedetectable change may be observed, after thawing the solution.

The rate of hybridization can also be increased by warming the solutioncontaining the nucleic acid to be detected and thenanoparticle-oligonucleotide conjugates to a temperature below thedissociation temperature (Tm) for the complex formed between theoligonucleotides on the nanoparticles and the target nucleic acid.Alternatively, rapid hybridization can be achieved by heating above thedissociation temperature (Tm) and allowing the solution to cool.

The rate of hybridization can also be increased by increasing the saltconcentration (e.g., from 0.1 M to 0.3 M NaCl).

The detectable change that occurs upon hybridization of theoligonucleotides on the nanoparticles to the nucleic acid may be a colorchange, the formation of aggregates of the nanoparticles, or theprecipitation of the aggregated nanoparticles. The color changes can beobserved with the naked eye or spectroscopically. The formation ofaggregates of the nanoparticles can be observed by electron microscopyor by nephelometry. The precipitation of the aggregated nanoparticlescan be observed with the naked eye or microscopically. Preferred arechanges observable with the naked eye. Particularly preferred is a colorchange observable with the naked eye.

The observation of a color change with the naked eye can be made morereadily against a background of a contrasting color. For instance, whengold nanoparticles are used, the observation of a color change isfacilitated by spotting a sample of the hybridization solution on asolid white surface (such as silica or alumina TLC plates, filter paper,cellulose nitrate membranes, and nylon membranes, preferably a C-18silica TLC plate) and allowing the spot to dry. Initially, the spotretains the color of the hybridization solution (which ranges frompink/red, in the absence of hybridization, to purplish-red/purple, ifthere has been hybridization). On drying at room temperature or 80° C.(temperature is not critical), a blue spot develops if thenanoparticle-oligonucleotide conjugates had been linked by hybridizationwith the target nucleic acid prior to spotting. In the absence ofhybridization (e.g., because no target nucleic acid is present), thespot is pink. The blue and the pink spots are stable and do not changeon subsequent cooling or heating or over time. They provide a convenientpermanent record of the test. No other steps (such as a separation ofhybridized and unhybridized nanoparticle-oligonucleotide conjugates) arenecessary to observe the color change.

An alternate method for easily visualizing the assay results is to spota sample of nanoparticle probes hybridized to a target nucleic acid on aglass fiber filter (e.g., Borosilicate Microfiber Filter, 0.7 micronpore size, grade FG75, for use with gold nanoparticles 13 nm in size),while drawing the liquid through the filter. Subsequent rinsing withwater washes the excess, non-hybridized probes through the filter,leaving behind an observable spot comprising the aggregates generated byhybridization of the nanoparticle probes with the target nucleic acid(retained because these aggregates are larger than the pores of thefilter). This technique may provide for greater sensitivity, since anexcess of nanoparticle probes can be used. Unfortunately, thenanoparticle probes stick to many other solid surfaces that have beentried (silica slides, reverse-phase plates, and nylon, nitrocellulose,cellulose and other membranes), and these surfaces cannot be used.

An important aspect of the detection system illustrated in FIG. 2 isthat obtaining a detectable change depends on cooperative hybridizationof two different oligonucleotides to a given target sequence in thenucleic acid. Mismatches in either of the two oligonucleotides willdestabilize the interparticle connection. It is well known that amismatch in base pairing has a much greater destabilizing effect on thebinding of a short oligonucleotide probe than on the binding of a longoligonucleotide probe. The advantage of the system illustrated in FIG. 2is that it utilizes the base discrimination associated with a longtarget sequence and probe (eighteen base-pairs in the exampleillustrated in FIG. 2), yet has the sensitivity characteristic of ashort oligonucleotide probe (nine base-pairs in the example illustratedin FIG. 2).

The target sequence of the nucleic acid may be contiguous, as in FIG. 2,or the two portions of the target sequence may be separated by a thirdportion which is not complementary to the oligonucleotides on thenanoparticles, as illustrated in FIG. 3. In the latter case, one has theoption of using a filler oligonucleotide which is free in solution andwhich has a sequence complementary to that of this third portion (seeFIG. 3). When the filler oligonucleotide hybridizes with the thirdportion of the nucleic acid, a double-stranded segment is created,thereby altering the average distance between the nanoparticles and,consequently, the color. The system illustrated in FIG. 3 may increasethe sensitivity of the detection method.

Some embodiments of the method of detecting nucleic acid utilize asubstrate. By employing a substrate, the detectable change (the signal)can be amplified and the sensitivity of the assay increased.

Any substrate can be used which allows observation of the detectablechange. Suitable substrates include transparent solid surfaces (e.g.,glass, quartz, plastics and other polymers), opaque solid surface (e.g.,white solid surfaces, such as TLC silica plates, filter paper, glassfiber filters, cellulose nitrate membranes, nylon membranes), andconducting solid surfaces (e.g., indium-tin-oxide (ITO)). The substratecan be any shape or thickness, but generally will be flat and thin.Preferred are transparent substrates such as glass (e.g., glass slides)or plastics (e.g., wells of microtiter plates).

In one embodiment, oligonucleotides are attached to the substrate. Theoligonucleotides can be attached to the substrates as described in,e.g., Chrisey et al., Nucleic Acids Res., 24, 3031–3039 (1996); Chriseyet al., Nucleic Acids Res., 24, 3040–3047 (1996); Mucic et al., Chem.Commun., 555 (1996); Zimmermann and Cox, Nucleic Acids Res., 22, 492(1994); Bottomley et al., J. Vac. Sci. Technol. A, 10, 591 (1992); andHegner et al., FEBS Lett., 336, 452 (1993).

The oligonucleotides attached to the substrate have a sequencecomplementary to a first portion of the sequence of a nucleic acid to bedetected. The nucleic acid is contacted with the substrate underconditions effective to allow hybridization of the oligonucleotides onthe substrate with the nucleic acid. In this manner the nucleic acidbecomes bound to the substrate. Any unbound nucleic acid is preferablywashed from the substrate before adding nanoparticle-oligonucleotideconjugates.

Next, the nucleic acid bound to the substrate is contacted with a firsttype of nanoparticles having oligonucleotides attached thereto. Theoligonucleotides have a sequence complementary to a second portion ofthe sequence of the nucleic acid, and the contacting takes place underconditions effective to allow hybridization of the oligonucleotides onthe nanoparticles with the nucleic acid. In this manner the first typeof nanoparticles become bound to the substrate. After thenanoparticle-oligonucleotide conjugates are bound to the substrate, thesubstrate is washed to remove any unbound nanoparticle-oligonucleotideconjugates and nucleic acid.

The oligonucleotides on the first type of nanoparticles may all have thesame sequence or may have different sequences that hybridize withdifferent portions of the nucleic acid to be detected. Whenoligonucleotides having different sequences are used, each nanoparticlemay have all of the different oligonucleotides attached to it or,preferably, the different oligonucleotides are attached to differentnanoparticles. FIG. 17 illustrates the use ofnanoparticle-oligonucleotide conjugates designed to hybridize tomultiple portions of a nucleic acid. Alternatively, the oligonucleotideson each of the first type of nanoparticles may have a plurality ofdifferent sequences, at least one of which must hybridize with a portionof the nucleic acid to be detected (see FIG. 25B).

Finally, the first type of nanoparticle-oligonucleotide conjugates boundto the substrate is contacted with a second type of nanoparticles havingoligonucleotides attached thereto. These oligonucleotides have asequence complementary to at least a portion of the sequence(s) of theoligonucleotides attached to the first type of nanoparticles, and thecontacting takes place under conditions effective to allow hybridizationof the oligonucleotides on the first type of nanoparticles with those onthe second type of nanoparticles. After the nanoparticles are bound, thesubstrate is preferably washed to remove any unboundnanoparticle-oligonucleotide conjugates.

The combination of hybridizations produces a detectable change. Thedetectable changes are the same as those described above, except thatthe multiple hybridizations result in an amplification of the detectablechange. In particular, since each of the first type of nanoparticles hasmultiple oligonucleotides (having the same or different sequences)attached to it, each of the first type of nanoparticle-oligonucleotideconjugates can hybridize to a plurality of the second type ofnanoparticle-oligonucleotide conjugates. Also, the first type ofnanoparticle-oligonucleotide conjugates may be hybridized to more thanone portion of the nucleic acid to be detected. The amplificationprovided by the multiple hybridizations may make the change detectablefor the first time or may increase the magnitude of the detectablechange. This amplification increases the sensitivity of the assay,allowing for detection of small amounts of nucleic acid.

If desired, additional layers of nanoparticles can be built up bysuccessive additions of the first and second types ofnanoparticle-oligonucleotide conjugates. In this way, the number ofnanoparticles immobilized per molecule of target nucleic acid can befurther increased with a corresponding increase in intensity of thesignal.

Also, instead of using first and second types ofnanoparticle-oligonucleotide conjugates designed to hybridize to eachother directly, nanoparticles bearing oligonucleotides that would serveto bind the nanoparticles together as a consequence of hybridizationwith binding oligonucleotides could be used.

Methods of making the nanoparticles and the oligonucleotides and ofattaching the oligonucleotides to the nanoparticles are described above.The hybridization conditions are well known in the art and can bereadily optimized for the particular system employed (see above).

An example of this method of detecting nucleic acid (analyte DNA) isillustrated in FIG. 13A. As shown in that Figure, the combination ofhybridizations produces dark areas where nanoparticle aggregates arelinked to the substrate by analyte DNA. These dark areas may be readilyobserved with the naked eye using ambient light, preferably viewing thesubstrate against a white background. As can be readily seen from FIG.13A, this method provides a means of amplifying a detectable change.

Another example of this method of detecting nucleic acid is illustratedin FIG. 25B. As in the example illustrated in FIG. 13A, the combinationof hybridizations produces dark areas where nanoparticle aggregates arelinked to the substrate by analyte DNA which can be observed with thenaked eye.

In another embodiment, nanoparticles are attached to the substrate.Nanoparticles can be attached to substrates as described in, e.g.,Grabar et al., Analyt. Chem., 67, 73–743 (1995); Bethell et al., J.Electroanal. Chem., 409, 137 (1996); Bar et al., Langmuir, 12, 1172(1996); Colvin et al., J. Am. Chem. Soc., 114, 5221 (1992).

After the nanoparticles are attached to the substrate, oligonucleotidesare attached to the nanoparticles. This may be accomplished in the samemanner described above for the attachment of oligonucleotides tonanoparticles in solution. The oligonucleotides attached to thenanoparticles have a sequence complementary to a first portion of thesequence of a nucleic acid.

The substrate is contacted with the nucleic acid under conditionseffective to allow hybridization of the oligonucleotides on thenanoparticles with the nucleic acid. In this manner the nucleic acidbecomes bound to the substrate. Unbound nucleic acid is preferablywashed from the substrate prior to adding furthernanoparticle-oligonucleotide conjugates.

Then, a second type of nanoparticles having oligonucleotides attachedthereto is provided. These oligonucleotides have a sequencecomplementary to a second portion of the sequence of the nucleic acid,and the nucleic acid bound to the substrate is contacted with the secondtype of nanoparticle-oligonucleotide conjugates under conditionseffective to allow hybridization of the oligonucleotides on the secondtype of nanoparticle-oligonucleotide conjugates with the nucleic acid.In this manner, the second type of nanoparticle-oligonucleotideconjugates becomes bound to the substrate. After the nanoparticles arebound, any unbound nanoparticle-oligonucleotide conjugates and nucleicacid are washed from the substrate. A change (e.g., color change) may bedetectable at this point.

The oligonucleotides on the second type of nanoparticles may all havethe same sequence or may have different sequences that hybridize withdifferent portions of the nucleic acid to be detected. Whenoligonucleotides having different sequences are used, each nanoparticlemay have all of the different oligonucleotides attached to it or,preferably, the different oligonucleotides may be attached to differentnanoparticles. See FIG. 17.

Next, a binding oligonucleotide having a selected sequence having atleast two portions, the first portion being complementary to at least aportion of the sequence of the oligonucleotides on the second type ofnanoparticles, is contacted with the second type ofnanoparticle-oligonucleotide conjugates bound to the substrate underconditions effective to allow hybridization of the bindingoligonucleotide to the oligonucleotides on the nanoparticles. In thismanner, the binding oligonucleotide becomes bound to the substrate.After the binding oligonucleotides are bound, unbound bindingoligonucleotides are washed from the substrate.

Finally, a third type of nanoparticles having oligonucleotides attachedthereto is provided. The oligonucleotides have a sequence complementaryto the sequence of a second portion of the binding oligonucleotide. Thenanoparticle-oligonucleotide conjugates are contacted with the bindingoligonucleotide bound to the substrate under conditions effective toallow hybridization of the binding oligonucleotide to theoligonucleotides on the nanoparticles. After the nanoparticles arebound, unbound nanoparticle-oligonucleotide conjugates are washed fromthe substrate.

The combination of hybridizations produces a detectable change. Thedetectable changes are the same as those described above, except thatthe multiple hybridizations result in an amplification of the detectablechange. In particular, since each of the second type of nanoparticleshas multiple oligonucleotides (having the same or different sequences)attached to it, each of the second type of nanoparticle-oligonucleotideconjugates can hybridize to a plurality of the third type ofnanoparticle-oligonucleotide conjugates (through the bindingoligonucleotide). Also, the second type of nanoparticle-oligonucleotideconjugates may be hybridized to more than one portion of the nucleicacid to be detected. The amplification provided by the multiplehybridizations may make the change detectable for the first time or mayincrease the magnitude of the detectable change. The amplificationincreases the sensitivity of the assay, allowing for detection of smallamounts of nucleic acid.

If desired, additional layers of nanoparticles can be built up bysuccessive additions of the binding oligonucleotides and second andthird types of nanoparticle-oligonucleotide conjugates. In this way, thenanoparticles immobilized per molecule of target nucleic acid can befurther increased with a corresponding increase in intensity of thesignal.

Also, the use of the binding oligonucleotide can be eliminated, and thesecond and third types of nanoparticle-oligonucleotide conjugates can bedesigned so that they hybridize directly to each other.

Methods of making the nanoparticles and the oligonucleotides and ofattaching the oligonucleotides to the nanoparticles are described above.The hybridization conditions are well known in the art and can bereadily optimized for the particular system employed (see above).

An example of this method of detecting nucleic acid (analyte DNA) isillustrated in FIG. 13B. As shown in that Figure, the combination ofhybridizations produces dark areas where nanoparticle aggregates arelinked to the substrate by analyte DNA. These dark areas may be readilyobserved with the naked eye as described above. As can be seen from FIG.13B, this embodiment of the method of the invention provides anothermeans of amplifying the detectable change.

Another amplification scheme employs liposomes. In this scheme,oligonucleotides are attached to a substrate. Suitable substrates arethose described above, and the oligonucleotides can be attached to thesubstrates as described above. For instance, where the substrate isglass, this can be accomplished by condensing the oligonucleotidesthrough phosphoryl or carboxylic acid groups to aminoalkyl groups on thesubstrate surface (for related chemistry see Grabar et al., Anal. Chem.,67, 735–743 (1995)).

The oligonucleotides attached to the substrate have a sequencecomplementary to a first portion of the sequence of the nucleic acid tobe detected. The nucleic acid is contacted with the substrate underconditions effective to allow hybridization of the oligonucleotides onthe substrate with the nucleic acid. In this manner the nucleic acidbecomes bound to the substrate. Any unbound nucleic acid is preferablywashed from the substrate before adding additional components of thesystem.

Next, the nucleic acid bound to the substrate is contacted withliposomes having oligonucleotides attached thereto. The oligonucleotideshave a sequence complementary to a second portion of the sequence of thenucleic acid, and the contacting takes place under conditions effectiveto allow hybridization of the oligonucleotides on the liposomes with thenucleic acid. In this manner the liposomes become bound to thesubstrate. After the liposomes are bound to the substrate, the substrateis washed to remove any unbound liposomes and nucleic acid.

The oligonucleotides on the liposomes may all have the same sequence ormay have different sequences that hybridize with different portions ofthe nucleic acid to be detected. When oligonucleotides having differentsequences are used, each liposome may have all of the differentoligonucleotides attached to it or the different oligonucleotides may beattached to different liposomes.

To prepare oligonucleotide-liposome conjugates, the oligonucleotides arelinked to a hydrophobic group, such as cholesteryl (see Letsinger etal., J. Am. Chem. Soc., 115, 7535–7536 (1993)), and thehydrophobic-oligonucleotide conjugates are mixed with a solution ofliposomes to form liposomes with hydrophobic-oligonucleotide conjugatesanchored in the membrane (see Zhang et al., Tetrahedron Lett., 37,6243–6246 (1996)). The loading of hydrophobic-oligonucleotide conjugateson the surface of the liposomes can be controlled by controlling theratio of hydrophobic-oligonucleotide conjugates to liposomes in themixture. It has been observed that liposomes bearing oligonucleotidesattached by hydrophobic interaction of pendent cholesteryl groups areeffective in targeting polynucleotides immobilized on a nitrocellulosemembrane (Id.). Fluorescein groups anchored in the membrane of theliposome were used as the reporter group. They served effectively, butsensitivity was limited by the fact that the signal from fluorescein inregions of high local concentration (e.g., on the liposome surface) isweakened by self quenching.

The liposomes are made by methods well known in the art. See Zhang etal., Tetrahedron Lett., 37, 6243 (1996). The liposomes will generally beabout 5–50 times larger in size (diameter) than the nanoparticles usedin subsequent steps. For instance, for nanoparticles about 13 nm indiameter, liposomes about 100 nm in diameter are preferably used.

The liposomes bound to the substrate are contacted with a first type ofnanoparticles having at least a first type of oligonucleotides attachedthereto. The first type of oligonucleotides have a hydrophobic groupattached to the end not attached to the nanoparticles, and thecontacting takes place under conditions effective to allow attachment ofthe oligonucleotides on the nanoparticles to the liposomes as a resultof hydrophobic interactions. A detectable change may be observable atthis point.

The method may further comprise contacting the first type ofnanoparticle-oligonucleotide conjugates bound to the liposomes with asecond type of nanoparticles having oligonucleotides attached thereto.The first type of nanoparticles have a second type of oligonucleotidesattached thereto which have a sequence complementary to at least aportion of the sequence of the oligonucleotides on the second type ofnanoparticles, and the oligonucleotides on the second type ofnanoparticles have a sequence complementary to at least a portion of thesequence of the second type of oligonucleotides on the first type ofnanoparticles. The contacting takes place under conditions effective toallow hybridization of the oligonucleotides on the first and secondtypes of nanoparticles. This hybridization will generally be performedat mild temperatures (e.g., 5° C. to 60° C.), so conditions (e.g.,0.3–1.0 M NaCl) conducive to hybridization at room temperature areemployed. Following hybridization, unbound nanoparticle-oligonucleotideconjugates are washed from the substrate.

The combination of hybridizations produces a detectable change. Thedetectable changes are the same as those described above, except thatthe multiple hybridizations result in an amplification of the detectablechange. In particular, since each of the liposomes has multipleoligonucleotides (having the same or different sequences) attached toit, each of the liposomes can hybridize to a plurality of the first typeof nanoparticle-oligonucleotide conjugates. Similarly, since each of thefirst type of nanoparticles has multiple oligonucleotides attached toit, each of the first type of nanoparticle-oligonucleotide conjugatescan hybridize to a plurality of the second type ofnanoparticle-oligonucleotide conjugates. Also, the liposomes may behybridized to more than one portion of the nucleic acid to be detected.The amplification provided by the multiple hybridizations may make thechange detectable for the first time or may increase the magnitude ofthe detectable change. This amplification increases the sensitivity ofthe assay, allowing for detection of small amounts of nucleic acid.

If desired, additional layers of nanoparticles can be built up bysuccessive additions of the first and second types ofnanoparticle-oligonucleotide conjugates. In this way, the number ofnanoparticles immobilized per molecule of target nucleic acid can befurther increased with a corresponding increase in the intensity of thesignal.

Also, instead of using second and third types ofnanoparticle-oligonucleotide conjugates designed to hybridize to eachother directly, nanoparticles bearing oligonucleotides that would serveto bring the nanoparticles together as a consequence of hybridizationwith binding oligonucleotides could be used.

Methods of making the nanoparticles and the oligonucleotides and ofattaching the oligonucleotides to the nanoparticles are described above.A mixture of oligonucleotides functionalized at one end for binding tothe nanoparticles and with or without a hydrophobic group at the otherend can be used on the first type of nanoparticles. The relative ratioof these oligonucleotides bound to the average nanoparticle will becontrolled by the ratio of the concentrations of the twooligonucleotides in the mixture. The hybridization conditions are wellknown in the art and can be readily optimized for the particular systememployed (see above).

An example of this method of detecting nucleic acid is illustrated inFIG. 18. The hybridization of the first type ofnanoparticle-oligonucleotide conjugates to the liposomes may produce adetectable change. In the case of gold nanoparticles, a pink/red colormay be observed or a purple/blue color may be observed if thenanoparticles are close enough together. The hybridization of the secondtype of nanoparticle-oligonucleotide conjugates to the first type ofnanoparticle-oligonucleotide conjugates will produce a detectablechange. In the case of gold nanoparticles, a purple/blue color will beobserved. All of these color changes may be observed with the naked eye.

In yet other embodiments utilizing a substrate, an “aggregate probe” canbe used. The aggregate probe can be prepared by allowing two types ofnanoparticles having complementary oligonucleotides (a and a′) attachedto them to hybridize to form a core (illustrated in FIG. 28A). Sinceeach type of nanoparticle has a plurality of oligonucleotides attachedto it, each type of nanoparticles will hybridize to a plurality of theother type of nanoparticles. Thus, the core is an aggregate containingnumerous nanoparticles of both types. The core is then capped with athird type of nanoparticles having at least two types ofoligonucleotides attached to them. The first type of oligonucleotideshas a sequence b which is complementary to the sequence b′ of a portionof a nucleic acid to be detected. The second type of oligonucleotideshas sequence a or a′ so that the third type of nanoparticles willhybridize to nanoparticles on the exterior of the core. The aggregateprobe can also be prepared by utilizing two types of nanoparticles (seeFIG. 28B). Each type of nanoparticles has at least two types ofoligonucleotides attached to them. The first type of oligonucleotidespresent on each of the two types of nanoparticles has sequence b whichis complementary to the sequence b′ of a portion of the nucleic acid tobe detected. The second type of oligonucleotides on the first type ofnanoparticles has a sequence a which is complementary to the sequence a′of the second type of oligonucleotides on the second type ofnanoparticles (see FIG. 28B) so that the two types of nanoparticleshybridize to each other to form the aggregate probe. Since each type ofnanoparticles has a plurality of oligonucleotides attached to it, eachtype of nanoparticles will hybridize to a plurality of the other type ofnanoparticles to form an aggregate containing numerous nanoparticles ofboth types.

The aggregate probe can be utilized to detect nucleic acid in any of theabove assay formats performed on a substrate, eliminating the need tobuild up layers of individual nanoparticles in order to obtain orenhance a detectable change. To even further enhance the detectablechange, layers of aggregate probes can be built up by using two types ofaggregate probes, the first type of aggregate probe havingoligonucleotides attached to it that are complementary tooligonucleotides on the other type of aggregate probe. In particular,when the aggregate probe is prepared as illustrated in FIG. 28B, theaggregate probes can hybridize to each other to form the multiplelayers. Some of the possible assay formats utilizing aggregate probesare illustrated in FIGS. 28C–D. For instance, a type of oligonucleotidescomprising sequence c is attached to a substrate (see FIG. 28C).Sequence c is complementary to the sequence c′ of a portion of a nucleicacid to be detected. The target nucleic acid is added and allowed tohybridize to the oligonucleotides attached to the substrate, after whichthe aggregate probe is added and allowed to hybridize to the portion ofthe target nucleic acid having sequence b′, thereby producing adetectable change. Alternatively, the target nucleic acid can first behybridized to the aggregate probe in solution and subsequentlyhybridized to the oligonucleotides on the substrate, or the targetnucleic acid can simultaneously be hybridized to the aggregate probe andthe oligonucleotides on the substrate. In another embodiment, the targetnucleic acid is allowed to react with the aggregate probe and anothertype of nanoparticles in solution (see FIG. 28D). Some of theoligonucleotides attached to this additional type of nanoparticlescomprise sequence c so that they hybridize to sequence c′ of the targetnucleic acid and some of the oligonucleotides attached to thisadditional type of nanoparticles comprise sequence d so that they cansubsequently hybridize to oligonucleotides comprising sequence d′ whichare attached to the substrate.

The core itself can also be used as a probe to detect nucleic acids. Onepossible assay format is illustrated in FIG. 28E. As illustrated there,a type of oligonucleotides comprising sequence b is attached to asubstrate. Sequence b is complementary to the sequence b′ of a portionof a nucleic acid to be detected. The target nucleic acid is contactedwith the substrate and allowed to hybridize to the oligonucleotidesattached to the substrate. Then, another type of nanoparticles is added.Some of the oligonucleotides attached to this additional type ofnanoparticles comprise sequence c so which is complementary to sequencec′ of the target nucleic acid so that the nanoparticles hybridize to thetarget nucleic acid bound to the substrate. Some of the oligonucleotidesattached to the additional type of nanoparticles comprise sequence a ora′ complementary to sequences a and a′ on the core probe, and the coreprobe is added and allowed to hybridize to the oligonucleotides on thenanoparticles. Since each core probe has sequences a and a′ attached tothe nanoparticles which comprise the core, the core probes can hybridizeto each other to form multiple layers attached to the substrate,providing a greatly enhanced detectable change. In alternativeembodiments, the target nucleic acid could be contacted with theadditional type of nanoparticles in solution prior to being contactedwith the substrate, or the target nucleic acid, the nanoparticles andthe substrate could all be contacted simultaneously. In yet anotheralternative embodiment, the additional type of nanoparticles could bereplaced by a linking oligonucleotide comprising both sequences c and aor a′.

When a substrate is employed, a plurality of the initial types ofnanoparticle-oligonucleotide conjugates or oligonucleotides can beattached to the substrate in an array for detecting multiple portions ofa target nucleic acid, for detecting multiple different nucleic acids,or both. For instance, a substrate may be provided with rows of spots,each spot containing a different type of oligonucleotide oroligonucleotide-nanoparticle conjugate designed to bind to a portion ofa target nucleic acid. A sample containing one or more nucleic acids isapplied to each spot, and the rest of the assay is performed in one ofthe ways described above using appropriate oligonucleotide-nanoparticleconjugates, oligonucleotide-liposome conjugates, aggregate probes, coreprobes, and binding oligonucleotides.

Finally, when a substrate is employed, a detectable change can beproduced or further enhanced by silver staining. Silver staining can beemployed with any type of nanoparticles that catalyze the reduction ofsilver. Preferred are nanoparticles made of noble metals (e.g., gold andsilver). See Bassell, et al., J. Cell Biol., 126, 863–876 (1994);Braun-Howland et al., Biotechniques, 13,928–931 (1992). If thenanoparticles being employed for the detection of a nucleic acid do notcatalyze the reduction of silver, then silver ions can be complexed tothe nucleic acid to catalyze the reduction. See Braun et al., Nature,391, 775 (1998). Also, silver stains are known which can react with thephosphate groups on nucleic acids.

Silver staining can be used to produce or enhance a detectable change inany assay performed on a substrate, including those described above. Inparticular, silver staining has been found to provide a huge increase insensitivity for assays employing a single type of nanoparticle, such asthe one illustrated in FIG. 25A, so that the use of layers ofnanoparticles, aggregate probes and core probes can often be eliminated.

In assays for detecting nucleic acids performed on a substrate, thedetectable change can be observed with an optical scanner. Suitablescanners include those used to scan documents into a computer which arecapable of operating in the reflective mode (e.g., a flatbed scanner),other devices capable of performing this function or which utilize thesame type of optics, any type of greyscale-sensitive measurement device,and standard scanners which have been modified to scan substratesaccording to the invention (e.g., a flatbed scanner modified to includea holder for the substrate) (to date, it has not been found possible touse scanners operating in the transmissive mode). The resolution of thescanner must be sufficient so that the reaction area on the substrate islarger than a single pixel of the scanner. The scanner can be used withany substrate, provided that the detectable change produced by the assaycan be observed against the substrate (e.g., a grey spot, such as thatproduced by silver staining, can be observed against a white background,but cannot be observed against a grey background). The scanner can be ablack-and-white scanner or, preferably, a color scanner. Mostpreferably, the scanner is a standard color scanner of the type used toscan documents into computers. Such scanners are inexpensive and readilyavailable commercially. For instance, an Epson Expression 636 (600×600dpi), a UMAX Astra 1200 (300×300 dpi), or a Microtec 1600 (1600×1600dpi) can be used. The scanner is linked to a computer loaded withsoftware for processing the images obtained by scanning the substrate.The software can be standard software which is readily availablecommercially, such as Adobe Photoshop 5.2 and Corel Photopaint 8.0.Using the software to calculate greyscale measurements provides a meansof quantitating the results of the assays. The software can also providea color number for colored spots and can generate images (e.g.,printouts) of the scans which can be reviewed to provide a qualitativedetermination of the presence of a nucleic acid, the quantity of anucleic acid, or both. In addition, it has been found that thesensitivity of assays such as that described in Example 5 can beincreased by subtracting the color that represents a negative result(red in Example 5) from the color that represents a positive result(blue in Example 5). The computer can be a standard personal computerwhich is readily available commercially. Thus, the use of a standardscanner linked to a standard computer loaded with standard software canprovide a convenient, easy, inexpensive means of detecting andquantitating nucleic acids when the assays are performed on substrates.The scans can also be stored in the computer to maintain a record of theresults for further reference or use. Of course, more sophisticatedinstruments and software can be used, if desired.

A nanoparticle-oligonucleotide conjugate which may be used in an assayfor any nucleic acid is illustrated in FIGS. 17D–E. This “universalprobe” has oligonucleotides of a single sequence attached to it. Theseoligonucleotides can hybridize with a binding oligonucleotide which hasa sequence comprising at least two portions. The first portion iscomplementary to at least a portion of the sequence of theoligonucleotides on the nanoparticles. The second portion iscomplementary to a portion of the sequence of the nucleic acid to bedetected. A plurality of binding oligonucleotides having the same firstportion and different second portions can be used, in which case the“universal probe”, after hybridization to the binding oligonucleotides,can bind to multiple portions of the nucleic acid to be detected or todifferent nucleic acid targets.

In a number of other embodiments of the invention, the detectable changeis created by labeling the oligonucleotides, the nanoparticles, or bothwith molecules (e.g., fluorescent molecules and dyes) that producedetectable changes upon hydridization of the oligonucleotides on thenanoparticles with the target nucleic acid. For instance,oligonucleotides attached to metal and semiconductor nanoparticles canhave a fluorescent molecule attached to the end not attached to thenanoparticles. Metal and semiconductor nanoparticles are knownfluorescence quenchers, with the magnitude of the quenching effectdepending on the distance between the nanoparticles and the fluorescentmolecule. In the unhybridized state, the oligonucleotides attached tothe nanoparticles interact with the nanoparticles, so that significantquenching will be observed. See FIG. 20A. Upon hybridization to a targetnucleic acid, the fluorescent molecule will become spaced away from thenanoparticles, diminishing quenching of the fluorescence. See FIG. 20A.Longer oligonucleotides should give rise to larger changes influorescence, at least until the fluorescent groups are moved far enoughaway from the nanoparticle surfaces so that an increase in the change isno longer observed. Useful lengths of the oligonucleotides can bedetermined empirically. Metallic and semiconductor nanoparticles havingfluorescent-labeled oligonucleotides attached thereto can be used in anyof the assay formats described above, including those performed insolution or on substrates.

Methods of labeling oligonucleotides with fluorescent molecules andmeasuring fluorescence are well known in the art. Suitable fluorescentmolecules are also well known in the art and include the fluoresceins,rhodamines and Texas Red. The oligonucleotides will be attached to thenanoparticles as described above.

In yet another embodiment, two types of fluorescent-labeledoligonucleotides attached to two different particles can be used.Suitable particles include polymeric particles (such as polystyreneparticles, polyvinyl particles, acrylate and methacrylate particles),glass particles, latex particles, Sepharose beads and others likeparticles well known in the art. Methods of attaching oligonucleotidesto such particles are well known in the art. See Chrisey et al., NucleicAcids Research, 24, 3031–3039 (1996) (glass) and Charreyre et al.,Langmuir, 13, 3103–3110 (1997), Fahy et al., Nucleic Acids Research, 21,1819–1826 (1993), Elaissari et al., J. Colloid Interface Sci., 202,251–260 (1998), Kolarova et al., Biotechniques, 20, 196–198 (1996) andWolf et al., Nucleic Acids Research, 15, 2911–2926 (1987)(polymer/latex). In particular, a wide variety of functional groups areavailable on the particles or can be incorporated into such particles.Functional groups include carboxylic acids, aldehydes, amino groups,cyano groups, ethylene groups, hydroxyl groups, mercapto groups, and thelike. Nanoparticles, including metallic and semiconductor nanoparticles,can also be used.

The two fluorophores are designated d and a for donor and acceptor. Avariety of fluorescent molecules useful in such combinations are wellknown in the art and are available from, e.g., Molecular Probes. Anattractive combination is fluorescein as the donor and Texas Red asacceptor. The two types of nanoparticle-oligonucleotide conjugates withd and a attached are mixed with the target nucleic acid, andfluorescence measured in a fluorimeter. The mixture will be excited withlight of the wavelength that excites d, and the mixture will bemonitored for fluorescence from a. Upon hybridization, d and a will bebrought in proximity (see FIG. 20B). In the case of non-metallic,non-semiconductor particles, hybridization will be shown by a shift influorescence from that for d to that for a or by the appearance offluorescence for a in addition to that for d. In the absence ofhybridization, the flurophores will be too far apart for energy transferto be significant, and only the fluorescence of d will be observed. Inthe case of metallic and semiconductor nanoparticles, lack ofhybridization will be shown by a lack of fluorescence due to d or abecause of quenching (see above). Hybridization will be shown by anincrease in fluorescence due to a.

As will be appreciated, the above described particles and nanoparticleshaving oligonucleotides labeled with acceptor and donor fluorescentmolecules attached can be used in the assay formats described above,including those performed in solution and on substrates. For solutionformats, the oligonucleotide sequences are preferably chosen so thatthey bind to the target nucleic acid as illustrated in FIGS. 15A–G. Inthe formats shown in FIG. 13A–B and 18, the binding oligonucleotides maybe used to bring the acceptor and donor fluorescent molecules on the twonanoparticles in proximity. Also, in the format illustrated in FIG. 13A,the oligonucleotides attached the substrate may be labeled with d.Further, other labels besides fluorescent molecules can be used, such aschemiluminescent molecules, which will give a detectable signal or achange in detectable signal upon hybridization.

Another embodiment of the detection method of the invention is a verysensitive system that utilizes detection of changes in fluorescence andcolor (illustrated in FIG. 21). This system employs latex microspheresto which are attached oligonucleotides labeled with a fluorescentmolecule and gold nanoparticles to which are attached oligonucleotides.The oligonucleotide-nanoparticle conjugates can be prepared as describedabove. Methods of attaching oligonucleotides to latex microspheres arewell known (see, e.g., Charreyre et al., Langmuir, 13:3103–3110 (1997);Elaissari et al., J. Colloid Interface Sci., 202:251–260 (1998)), as aremethods of labeling oligonucleotides with fluorescent molecules (seeabove). The oligonucleotides on the latex microspheres and theoligonucleotides on the gold nanoparticles have sequences capable ofhybridizing with different portions of the sequence of a target nucleicacid, but not with each other. When a target nucleic acid comprisingsequences complementary to the sequences of the oligonucleotides or thelatex microspheres and gold nanoparticles is contacted with the twoprobes, a network structure is formed (see FIG. 21). Due to thequenching properties of the gold nanoparticles, the fluorescence of theoligonucleotides attached to the latex microspheres is quenched whilepart of this network. Indeed, one gold nanoparticle can quench manyfluorophore molecules since gold nanoparticles have very largeabsorption coefficients. Thus, the fluorescence of a solution containingnucleic acid and the two particles can be monitored to detect theresults, with a reduction in, or elimination of, fluorescence indicatinga positive result. Preferably, however, the results of the assay aredetected by placing a droplet of the solution onto a microporousmaterial (see FIG. 21). The microporous material should be transparentor a color (e.g., white) which allows for detection of the pink/redcolor of the gold nanoparticles. The microporous material should alsohave a pore size sufficiently large to allow the gold nanoparticles topass through the pores and sufficiently small to retain the latexmicrospheres on the surface of the microporous material when themicroporous material is washed. Thus, when using such a microporousmaterial, the size (diameter) of the latex microspheres must be largerthan the size (diameter) of the gold nanoparticles. The microporousmaterial must also be inert to biological media. Many suitablemicroporous materials are known in the art and include various filtersand membranes, such as modified polyvinylidene fluoride (PVDF, such asDurapore™ membrane filters purchased from Millipore Corp.) and purecellulose acetate (such as AcetatePlus™ membrane filters purchased fromMicron Separations Inc.). Such a microporous material retains thenetwork composed of target nucleic acid and the two probes, and apositive result (presence of the target nucleic acid) is evidenced by ared/pink color (due to the presence of the gold nanoparticles) and alack of fluorescence (due to quenching of fluorescence by the goldnanoparticles) (see FIG. 21). A negative result (no target nucleic acidpresent) is evidenced by a white color and fluorescence, because thegold nanoparticles would pass through the pores of the microporousmaterial when it is washed (so no quenching of the fluorescence wouldoccur), and the white latex microspheres would be trapped on top of it(see FIG. 21). In addition, in the case of a positive result, changes influorescence and color can be observed as a function of temperature. Forinstance, as the temperature is raised, fluorescence will be observedonce the dehybridization temperature has been reached. Therefore, bylooking at color or fluorescence as a function of temperature,information can be obtained about the degree of complementarity betweenthe oligonucleotide probes and the target nucleic acid. As noted above,this detection method exhibits high sensitivity. As little as 3femtomoles of single-stranded target nucleic acid 24 bases in length and20 femtomoles of double-stranded target nucleic acid 24 bases in lengthhave been detected with the naked eye. The method is also very simple touse. Fluorescence can be generated by simply illuminating the solutionor microporous material with a UV lamp, and the fluorescent andcalorimetric signals can be monitored by the naked eye. Alternatively,for a more quantitative result, a fluorimeter can be employed infront-face mode to measure the fluorescence of the solution with a shortpathlength.

The above embodiment has been described with particular reference tolatex microspheres and gold nanoparticles. Any other microsphere ornanoparticle, having the other properties described above and to whicholigonucleotides can be attached, can be used in place of theseparticles. Many suitable particles and nanoparticles are describedabove, along with techniques for attaching oligonucleotides to them. Inaddition, microspheres and nanoparticles having other measurableproperties may be used. For instance, polymer-modified particles andnanoparticles, where the polymer can be modified to have any desirableproperty, such as fluorescence, color, or electrochemical activity, canbe used. See, Watson et al., J. Am. Chem. Soc., 121, 462463 (1999)(polymer-modified gold nanoparticles). Also, magnetic, polymer-coatedmagnetic, and semiconducting particles can be used. See Chan et al.,Science, 281, 2016 (1998); Bruchez et al., Science, 281, 2013 (1998);Kolarova et al., Biotechniques, 20, 196–198 (1996).

In yet another embodiment, two probes comprising metallic orsemiconductor nanoparticles having oligonucleotides labeled withfluorescent molecules attached to them are employed (illustrated in FIG.22). The oligonucleotide-nanoparticle conjugates can be prepared andlabeled with fluorescent molecules as described above. Theoligonucleotides on the two types of oligonucleotide-nanoparticleconjugates have sequences capable of hybridizing with different portionsof the sequence of a target nucleic acid, but not with each other. Whena target nucleic acid comprising sequences complementary to thesequences of the oligonucleotides on the nanoparticles is contacted withthe two probes, a network structure is formed (see FIG. 22). Due to thequenching properties of the metallic or semiconductor nanoparticles, thefluorescence of the oligonucleotides attached to the nanoparticles isquenched while part of this network. Thus, the fluorescence of asolution containing nucleic acid and the two probes can be monitored todetect the results, with a reduction in, or elimination of, fluorescenceindicating a positive result. Preferably, however, the results of theassay are detected by placing a droplet of the solution onto amicroporous material (see FIG. 22). The microporous material should havea pore size sufficiently large to allow the nanoparticles to passthrough the pores and sufficiently small to retain the network on thesurface of the microporous material when the microporous material iswashed (see FIG. 22). Many suitable microporous materials are known inthe art and include those described above. Such a microporous materialretains the network composed of target nucleic acid and the two probes,and a positive result (presence of the target nucleic acid) is evidencedby a lack of fluorescence (due to quenching of fluorescence by themetallic or semiconductor nanoparticles) (see FIG. 22). A negativeresult (no target nucleic acid present) is evidenced by fluorescencebecause the nanoparticles would pass through the pores of themicroporous material when it is washed (so no quenching of thefluorescence would occur) (see FIG. 22). There is low backgroundfluorescence because unbound probes are washed away from the detectionarea. In addition, in the case of a positive result, changes influorescence can be observed as a function of temperature. For instance,as the temperature is raised, fluorescence will be observed once thedehybridization temperature has been reached. Therefore, by looking atfluorescence as a function of temperature, information can be obtainedabout the degree of complementarity between the oligonucleotide probesand the target nucleic acid. Fluorescence can be generated by simplyilluminating the solution or microporous material with a UV lamp, andthe fluorescent signal can be monitored by the naked eye. Alternatively,for a more quantitative result, a fluorimeter can be employed infront-face mode to measure the fluorescence of the solution with a shortpath length.

In yet other embodiments, a “satellite probe” is used (see FIG. 24). Thesatellite probe comprises a central particle with one or severalphysical properties that can be exploited for detection in an assay fornucleic acids (e.g., intense color, fluorescence quenching ability,magnetism). Suitable particles include the nanoparticles and otherparticles described above. The particle has oligonucleotides (all havingthe same sequence) attached to it (see FIG. 24). Methods of attachingoligonucleotides to the particles are described above. Theseoligonucleotides comprise at least a first portion and a second portion,both of which are complementary to portions of the sequence of a targetnucleic acid (see FIG. 24). The satellite probe also comprises probeoligonucleotides. Each probe oligonucleotide has at least a firstportion and a second portion (see FIG. 24). The sequence of the firstportion of the probe oligonucleotides is complementary to the firstportion of the sequence of the oligonucleotides immobilized on thecentral particle (see FIG. 24). Consequently, when the central particleand the probe oligonucleotides are brought into contact, theoligonucleotides on the particle hybridize with the probeoligonucleotides to form the satellite probe (see FIG. 24). Both thefirst and second portions of the probe oligonucleotides arecomplementary to portions of the sequence of the target nucleic acid(see FIG. 24). Each probe oligonucleotide is labeled with a reportermolecule (see FIG. 24), as further described below. The amount ofhybridization overlap between the probe oligonucleotides and the target(length of the portion hybridized) is as large as, or greater than, thehybridization overlap between the probe oligonucleotides and theoligonucleotides attached to the particle (see FIG. 24). Therefore,temperature cycling resulting in dehybridization and rehybridizationwould favor moving the probe oligonucleotides from the central particleto the target. Then, the particles are separated from the probeoligonucleotides hybridized to the target, and the reporter molecule isdetected.

The satellite probe can be used in a variety of detection strategies.For example, if the central particle has a magnetic core and is coveredwith a material capable of quenching the fluorescence of fluorophoresattached to the probe oligonucleotides that surround it, this system canbe used in an in situ fluorometric detection scheme for nucleic acids.Functionalized polymer-coated magnetic particles (Fe₃O₄) are availablefrom several commercial sources including Dynal (Dynabeads™) and BangsLaboratories (Estapor™), and silica-coated magnetic Fe₃O₄ nanoparticlescould be modified (Liu et al., Chem. Mater., 10, 3936–3940 (1998)) usingwell-developed silica surface chemistry (Chrisey et al., Nucleic AcidsResearch, 24, 3031–3039 (1996)) and employed as magnetic probes as well.Further, the dye molecule, 4-((4-(dimethylamino)phenyl)-azo)benzoic acid(DABCYL) has been shown to be an efficient quencher of fluorescence fora wide variety of fluorphores attached to oligonucleotides (Tyagi etal., Nature Biotech., 16, 49–53 (1998). The commercially-availablesuccinimidyl ester of DABCYL (Molecular Probes) forms extremely stableamide bonds upon reaction with primary alkylamino groups. Thus, anymagnetic particle or polymer-coated magnetic particle with primary alkylamino groups could be modified with both oligonucleotides, as well asthese quencher molecules. Alternatively, the DABCYL quencher could beattached directly to the surface-bound oligonucleotide, instead of thealkyl amino-modified surface. The satellite probe comprising the probeoligonucleotides is brought into contact with the target. Thetemperature is cycled so as to cause dehybridization andrehybridization, which causes the probe oligonucleotides to move fromthe central particle to the target. Detection is accomplished byapplying a magnetic field and removing the particles from solution andmeasuring the fluorescence of the probe oligonucleotides remaining insolution hybridized to the target.

This approach can be extended to a colorimetric assay by using magneticparticles with a dye coating in conjunction with probe oligonucleotideslabeled with a dye which has optical properties that are distinct fromthe dye on the magnetic nanoparticles or perturb those of the dye on themagnetic nanoparticles. When the particles and the probeoligonucleotides are in solution together, the solution will exhibit onecolor which derives from a combination of the two dyes. However, in thepresence of a target nucleic acid and with temperature cycling, theprobe oligonucleotides will move from the satellite probe to the target.Once this has happened, application of a magnetic field will remove themagnetic, dye-coated particles from solution leaving behind probeoligonucleotides labeled with a single dye hybridized to the target. Thesystem can be followed with a colorimeter or the naked eye, dependingupon target levels and color intensities.

This approach also can be further extended to an electrochemical assayby using an oligonucleotide-magnetic particle conjugate in conjunctionwith a probe oligonucleotide having attached a redox-active molecule.Any modifiable redox-active species can be used, such as thewell-studied redox-active ferrocene derivative. A ferrocene derivatizedphosphoramidite can be attached to oligonucleotides directly usingstandard phosphoramidite chemistry. Mucic et al., Chem. Commun., 555(1996); Eckstein, ed., in Oligonucleotides and Analogues, 1st ed.,Oxford University, New York, N.Y. (1991). The ferrocenylphosphoramiditeis prepared in a two-step synthesis from 6-bromohexylferrocene. In atypical preparation, 6-bromohexylferrocene is stirred in an aqueous HMPAsolution at 120° C. for 6 hours to from 6-hydroxyhexylferrocene. Afterpurification, the 6-hydroxyhexylferrocene is added to a THF solution ofN,N-diisopropylethylamine andbeta-cyanoethyl-N,N-diisopropylchlorophosphoramide to form theferrocenylphosphoramidite. Oligonucleotide-modified polymer-coated goldnanoparticles, where the polymer contains electrochemically-activeferrocene molecules, could also be utilized. Watson et al., J. Am. Chem.Soc., 121, 462–463 (1999). A copolymer of amino reactive sites (e.g.,anhydrides) could be incorporated into the polymer for reaction withamino-modified oligonucleotides. Moller et al., Bioconjugate Chem., 6,174–178 (1995). In the presence of target and with temperature cycling,the redox-active probe oligonucleotides will move from the satelliteprobe to the target. Once this has happened, application of the magneticfield will remove the magnetic particles from solution leaving behindthe redox-active probe oligonucleotides hybridized with the targetnucleic acid. The amount of target then can be determined by cyclicvoltammetry or any electrochemical technique that can interrogate theredox-active molecule.

In yet another embodiment of the invention, a nucleic acid is detectedby contacting the nucleic acid with a substrate having oligonucleotidesattached thereto. The oligonucleotides have a sequence complementary toa first portion of the sequence of the nucleic acid. Theoligonucleotides are located between a pair of electrodes located on thesubstrate. The substrate must be made of a material which is not aconductor of electricity (e.g., glass, quartz, polymers, plastics). Theelectrodes may be made of any standard material (e.g., metals, such asgold, platinum, tin oxide). The electrodes can be fabricated byconventional microfabrication techniques. See, e.g., Introduction ToMicrolithography (L. F. Thompson et al., eds., American ChemicalSociety, Washington, D.C. 1983). The substrate may have a plurality ofpairs of electrodes located on it in an array to allow for the detectionof multiple portions of a single nucleic acid, the detection of multipledifferent nucleic acids, or both. Arrays of electrodes can be purchased(e.g., from Abbtech Scientific, Inc., Richmond, Va.) or can be made byconventional microfabrication techniques. See, e.g., Introduction ToMicrolithography (L. F. Thompson et al., eds., American ChemicalSociety, Washington, D.C. 1983). Suitable photomasks for making thearrays can be purchased (e.g., from Photronics, Milpitas, Calif.). Eachof the pairs of electrodes in the array will have a type ofoligonucleotides attached to the substrate between the two electrodes.The contacting takes place under conditions effective to allowhybridization of the oligonucleotides on the substrate with the nucleicacid. Then, the nucleic acid bound to the substrate, is contacted with atype of nanoparticles. The nanoparticles must be made of a materialwhich can conduct electricity. Such nanoparticles include those made ofmetal, such as gold nanoparticles, and semiconductor materials. Thenanoparticles will have one or more types of oligonucleotides attachedto them, at least one of the types of oligonucleotides having a sequencecomplementary to a second portion of the sequence of the nucleic acid.The contacting takes place under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles with thenucleic acid. If the nucleic acid is present, the circuit between theelectrodes should be closed because of the attachment of thenanoparticles to the substrate between the electrodes, and a change inconductivity will be detected. If the binding of a single type ofnanoparticles does not result in closure of the circuit, this situationcan be remedied by using a closer spacing between the electrodes, usinglarger nanoparticles, or employing another material that will close thecircuit (but only if the nanoparticles have been bound to the substratebetween the electrodes). For instance, when gold nanoparticles are used,the substrate can be contacted with silver stain (as described above) todeposit silver between the electrodes to close the circuit and producethe detectable change in conductivity. Another way to close the circuitin the case where the addition of a single type of nanoparticles is notsufficient, is to contact the first type of nanoparticles bound to thesubstrate with a second type of nanoparticles having oligonucleotidesattached to them that have a sequence complementary to theoligonucleotides on the first type of nanoparticles. The contacting willtake place under conditions effective so that the oligonucleotides onthe second type of nanoparticle hybridize to those on the first type ofoligonucleotides. If needed, or desired, additional layers ofnanoparticles can be built up by alternately adding the first and secondtypes of nanoparticles until a sufficient number of nanoparticles areattached to the substrate to close the circuit. Another alternative tobuilding up individual layers of nanoparticles would be the use of anaggregate probe (see above).

The invention also provides kits for detecting nucleic acids. In oneembodiment, the kit comprises at least one container, the containerholding at least two types of nanoparticles having oligonucleotidesattached thereto. The oligonucleotides on the first type ofnanoparticles have a sequence complementary to the sequence of a firstportion of a nucleic acid. The oligonucleotides on the second type ofnanoparticles have a sequence complementary to the sequence of a secondportion of the nucleic acid. The container may further comprise filleroligonucleotides having a sequence complementary to a third portion ofthe nucleic acid, the third portion being located between the first andsecond portions. The filler oligonucleotide may also be provided in aseparate container.

In a second embodiment, the kit comprises at least two containers. Thefirst container holds nanoparticles having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a firstportion of a nucleic acid. The second container holds nanoparticleshaving oligonucleotides attached thereto which have a sequencecomplementary to the sequence of a second portion of the nucleic acid.The kit may further comprise a third container holding a filleroligonucleotide having a sequence complementary to a third portion ofthe nucleic acid, the third portion being located between the first andsecond portions.

In another alternative embodiment, the kits can have theoligonucleotides and nanoparticles in separate containers, and theoligonucleotides would have to be attached to the nanoparticles prior toperforming an assay to detect a nucleic acid. The oligonucleotidesand/or the nanoparticles may be functionalized so that theoligonucleotides can be attached to the nanoparticles. Alternatively,the oligonucleotides and/or nanoparticles may be provided in the kitwithout functional groups, in which case they must be functionalizedprior to performing the assay.

In another embodiment, the kit comprises at least one container. Thecontainer holds metallic or semiconductor nanoparticles havingoligonucleotides attached thereto. The oligonucleotides have a sequencecomplementary to a portion of a nucleic acid and have fluorescentmolecules attached to the ends of the oligonucleotides not attached tothe nanoparticles.

In yet another embodiment, the kit comprises a substrate, the substratehaving attached thereto nanoparticles. The nanoparticles haveoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid. The kit also includesa first container holding nanoparticles having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a secondportion of the nucleic acid. The oligonucleotides may have the same ordifferent sequences, but each of the oligonucleotides has a sequencecomplementary to a portion of the nucleic acid. The kit further includesa second container holding a binding oligonucleotide having a selectedsequence having at least two portions, the first portion beingcomplementary to at least a portion of the sequence of theoligonucleotides on the nanoparticles in the first container. The kitalso includes a third container holding nanoparticles havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to the sequence of a second portion of thebinding oligonucleotide.

In another embodiment, the kit comprises a substrate havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid. The kit also includesa first container holding nanoparticles having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a secondportion of the nucleic acid. The oligonucleotides may have the same ordifferent sequences, but each of the oligonucleotides has a sequencecomplementary to a portion of the nucleic acid. The kit further includesa second container holding nanoparticles having oligonucleotidesattached thereto which have a sequence complementary to at least aportion of the oligonucleotides attached to the nanoparticles in thefirst container.

In yet another embodiment, the kits can have the substrate,oligonucleotides and nanoparticles in separate containers. Thesubstrate, oligonucleotides, and nanoparticles would have to beappropriately attached to each other prior to performing an assay todetect a nucleic acid. The substrate, oligonucleotides and/or thenanoparticles may be functionalized to expedite this attachment.Alternatively, the substrate, oligonucleotides and/or nanoparticles maybe provided in the kit without functional groups, in which case theymust be functionalized prior to performing the assay.

In a further embodiment, the kit comprises a substrate havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid. The kit also includesa first container holding liposomes having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a secondportion of the nucleic acid and a second container holding nanoparticleshaving at least a first type of oligonucleotides attached thereto, thefirst type of oligonucleotides having a cholesteryl group attached tothe end not attached to the nanoparticles so that the nanoparticles canattach to the liposomes by hydrophobic interactions. The kit may furthercomprise a third container holding a second type of nanoparticles havingoligonucleotides attached thereto, the oligonucleotides having asequence complementary to at least a portion of the sequence of a secondtype of oligonucleotides attached to the first type of nanoparticles.The second type of oligonucleotides attached to the first type ofnanoparticles having a sequence complementary to the sequence of theoligonucleotides on the second type of nanoparticles.

In another embodiment, the kit may comprise a substrate havingnanoparticles attached to it. The nanoparticles have oligonucleotidesattached to them which have a sequence complementary to the sequence ofa first portion of a nucleic acid. The kit also includes a firstcontainer holding an aggregate probe. The aggregated probe comprises atleast two types of nanoparticles having oligonucleotides attached tothem. The nanoparticles of the aggregate probe are bound to each otheras a result of the hybridization of some of the oligonucleotidesattached to each of them. At least one of the types of nanoparticles ofthe aggregate probe has oligonucleotides attached to it which have asequence complementary to a second portion of the sequence of thenucleic acid.

In yet another embodiment, the kit may comprise a substrate havingoligonucleotides attached to it. The oligonucleotides have a sequencecomplementary to the sequence of a first portion of a nucleic acid. Thekit further includes a first container holding an aggregate probe. Theaggregate probe comprises at least two types of nanoparticles havingoligonucleotides attached to them. The nanoparticles of the aggregateprobe are bound to each other as a result of the hybridization of someof the oligonucleotides attached to each of them. At least one of thetypes of nanoparticles of the aggregate probe has oligonucleotidesattached thereto which have a sequence complementary to a second portionof the sequence of the nucleic acid.

In an additional embodiment, the kit may comprise a substrate havingoligonucleotides attached to it and a first container holding anaggregate probe. The aggregate probe comprises at least two types ofnanoparticles having oligonucleotides attached to them. Thenanoparticles of the aggregate probe are bound to each other as a resultof the hybridization of some of the oligonucleotides attached to each ofthem. At least one of the types of nanoparticles of the aggregate probehas oligonucleotides attached to it which have a sequence complementaryto a first portion of the sequence of the nucleic acid. The kit alsoincludes a second container holding nanoparticles. The nanoparticleshave at least two types of oligonucleotides attached to them. The firsttype of oligonucleotides has a sequence complementary to a secondportion of the sequence of the nucleic acid. The second type ofoligonucleotides has a sequence complementary to at least a portion ofthe sequence of the oligonucleotides attached to the substrate.

In another embodiment, the kit may comprise a substrate which hasoligonucleotides attached to it. The oligonucleotides have a sequencecomplementary to the sequence of a first portion of a nucleic acid. Thekit also comprises a first container holding liposomes havingoligonucleotides attached to them. The oligonucleotides have a sequencecomplementary to the sequence of a second portion of the nucleic acid.The kit further includes a second container holding an aggregate probecomprising at least two types of nanoparticles having oligonucleotidesattached to them. The nanoparticles of the aggregate probe are bound toeach other as a result of the hybridization of some of theoligonucleotides attached to each of them. At least one of the types ofnanoparticles of the aggregate probe has oligonucleotides attached to itwhich have a hydrophobic groups attached to the ends not attached to thenanoparticles.

In a further embodiment, the kit may comprise a first container holdingnanoparticles having oligonucleotides attached thereto. The kit alsoincludes one or more additional containers, each container holding abinding oligonucleotide. Each binding oligonucleotide has a firstportion which has a sequence complementary to at least a portion of thesequence of oligonucleotides on the nanoparticles and a second portionwhich has a sequence complementary to the sequence of a portion of anucleic acid to be detected. The sequences of the second portions of thebinding oligonucleotides may be different as long as each sequence iscomplementary to a portion of the sequence of the nucleic acid to bedetected.

In another embodiment, the kit comprises a container holding one type ofnanoparticles having oligonucleotides attached thereto and one or moretypes of binding oligonucleotides. Each of the types of bindingoligonucleotides has a sequence comprising at least two portions. Thefirst portion is complementary to the sequence of the oligonucleotideson the nanoparticles, whereby the binding oligonucleotides arehybridized to the oligonucleotides on the nanoparticles in thecontainer(s). The second portion is complementary to the sequence of aportion of the nucleic acid.

In another embodiment, kits may comprise one or two containers holdingtwo types of particles. The first type of particles havingoligonucleotides attached thereto which have a sequence complementary tothe sequence of a first portion of a nucleic acid. The oligonucleotidesare labeled with an energy donor on the ends not attached to theparticles. The second type of particles having oligonucleotides attachedthereto which have a sequence complementary to the sequence of a secondportion of a nucleic acid. The oligonucleotides are labeled with anenergy acceptor on the ends not attached to the particles. The energydonors and acceptors may be fluorescent molecules.

In a further embodiment, the kit comprises a first container holding atype of latex microspheres having oligonucleotides attached thereto. Theoligonucleotides have a sequence complementary to a first portion of thesequence of a nucleic acid and are labeled with a fluorescent molecule.The kit also comprises a second container holding a type of goldnanoparticles having oligonucleotides attached thereto. Theseoligonucleotides have a sequence complementary to a second portion ofthe sequence of the nucleic acid.

In another embodiment, the kit comprises a first container holding afirst type of metallic or semiconductor nanoparticles havingoligonucleotides attached thereto. The oligonucleotides have a sequencecomplementary to a first portion of the sequence of a nucleic acid andare labeled with a fluorescent molecule. The kit also comprises a secondcontainer holding a second type of metallic or semiconductornanoparticles having oligonucleotides attached thereto. Theseoligonucleotides have a sequence complementary to a second portion ofthe sequence of a nucleic acid and are labeled with a fluorescentmolecule.

In a further embodiment, the kit comprises a container holding asatellite probe. The satellite probe comprises a particle havingattached thereto oligonucleotides. The oligonucleotides have a firstportion and a second portion, both portions having sequencescomplementary to portions of the sequence of a nucleic acid. Thesatellite probe also comprises probe oligonucleotides hybridized to theoligonucleotides attached to the nanoparticles. The probeoligonucleotides have a first portion and a second portion. The firstportion has a sequence complementary to the sequence of the firstportion of the oligonucleotides attached to the particles, and bothportions have sequences complementary to portions of the sequence of thenucleic acid. The probe oligonucleotides also have a reporter moleculeattached to one end.

In another embodiment, the kit may comprise a container holding anaggregate probe. The aggregate probe comprises at least two types ofnanoparticles having oligonucleotides attached to them. Thenanoparticles of the aggregate probe are bound to each other as a resultof the hybridization of some of the oligonucleotides attached to each ofthem. At least one of the types of nanoparticles of the aggregate probehas oligonucleotides attached to it which have a sequence complementaryto a portion of the sequence of a nucleic acid.

In an additional embodiment, the kit may comprise a container holding anaggregate probe. The aggregate probe comprises at least two types ofnanoparticles having oligonucleotides attached to them. Thenanoparticles of the aggregate probe are bound to each other as a resultof the hybridization of some of the oligonucleotides attached to each ofthem. At least one of the types of nanoparticles of the aggregate probehas oligonucleotides attached to it which have a hydrophobic groupattached to the end not attached to the nanoparticles.

In yet another embodiment, the invention provides a kit comprising asubstrate having located thereon at least one pair of electrodes witholigonucleotides attached to the substrate between the electrodes. In apreferred embodiment, the substrate has a plurality of pairs ofelectrodes attached to it in an array to allow for the detection ofmultiple portions of a single nucleic acid, the detection of multipledifferent nucleic acids, or both.

The kits may also contain other reagents and items useful for detectingnucleic acid. The reagents may include PCR reagents, reagents for silverstaining, hybridization reagents, buffers, etc. Other items which may beprovided as part of the kit include a solid surface (for visualizinghybridization) such as a TLC silica plate, microporous materials,syringes, pipettes, cuvettes, containers, and a thermocycler (forcontrolling hybridization and dehybridization temperatures). Reagentsfor functionalizing the nucleotides or nanoparticles may also beincluded in the kit.

The precipitation of aggregated nanoparticles provides a means ofseparating a selected nucleic acid from other nucleic acids. Thisseparation may be used as a step in the purification of the nucleicacid. Hybridization conditions are those described above for detecting anucleic acid. If the temperature is below the Tm (the temperature atwhich one-half of an oligonucleotide is bound to its complementarystrand) for the binding of the oligonucleotides on the nanoparticles tothe nucleic acid, then sufficient time is needed for the aggregate tosettle. The temperature of hybridization (e.g., as measured by Tm)varies with the type of salt (NaCl or MgCl₂) and its concentration. Saltcompositions and concentrations are selected to promote hybridization ofthe oligonucleotides on the nanoparticles to the nucleic acid atconvenient working temperatures without inducing aggregation of thecolloids in the absence of the nucleic acid.

The invention also provides a method of nanofabrication. The methodcomprises providing at least one type of linking oligonucleotide havinga selected sequence. A linking oligonucleotide used for nanofabricationmay have any desired sequence and may be single-stranded ordouble-stranded. It may also contain chemical modifications in the base,sugar, or backbone sections. The sequences chosen for the linkingoligonucleotides and their lengths and strandedness will contribute tothe rigidity or flexibility of the resulting nanomaterial ornanostructure, or a portion of the nanomaterial or nanostructure. Theuse of a single type of linking oligonucleotide, as well as mixtures oftwo or more different types of linking oligonucleotides, iscontemplated. The number of different linking oligonucleotides used andtheir lengths will contribute to the shapes, pore sizes and otherstructural features of the resulting nanomaterials and nanostructures.

The sequence of a linking oligonucleotide will have at least a firstportion and a second portion for binding to oligonucleotides onnanoparticles. The first, second or more binding portions of the linkingoligonucleotide may have the same or different sequences.

If all of the binding portions of a linking oligonucleotide have thesame sequence, only a single type of nanoparticle with oligonucleotideshaving a complementary sequence attached thereto need be used to form ananomaterial or nanostructure. If the two or more binding portions of alinking oligonucleotide have different sequences, then two or morenanoparticle-oligonucleotide conjugates must be used. See, e.g., FIG.17. The oligonucleotides on each of the nanoparticles will have asequence complementary to one of the two or more binding portions of thesequence of the linking oligonucleotide The number, sequence(s) andlength(s) of the binding portions and the distance(s), if any, betweenthem will contribute to the structural and physical properties of theresulting nanomaterials and nanostructures. Of course, if the linkingoligonucleotide comprises two or more portions, the sequences of thebinding portions must be chosen so that they are not complementary toeach other to avoid having one portion of the linking nucleotide bind toanother portion.

The linking oligonucleotides and nanoparticle-oligonucleotide conjugatesare contacted under conditions effective for hybridization of theoligonucleotides attached to the nanoparticles with the linkingoligonucleotides so that a desired nanomaterial or nanostructure isformed wherein the nanoparticles are held together by oligonucleotideconnectors. These hybridization conditions are well known in the art andcan be optimized for a particular nanofabrication scheme (see above).Stringent hybridization conditions are preferred.

The invention also provides another method of nanofabrication. Thismethod comprises providing at least two types ofnanoparticle-oligonucleotide conjugates. The oligonucleotides on thefirst type of nanoparticles have a sequence complementary to that of theoligonucleotides on the second type of nanoparticles. Theoligonucleotides on the second type of nanoparticles have a sequencecomplementary to that of the oligonucleotides on the first type ofnanoparticles. The nanoparticle-oligonucleotide conjugates are contactedunder conditions effective to allow hybridization of theoligonucleotides on the nanoparticles to each other so that a desirednanomaterial or nanostructure is formed wherein the nanoparticles areheld together by oligonucleotide connectors. Again, these hybridizationconditions are well-known in the art and can be optimized for aparticular nanofabrication scheme.

In both nanofabrication methods of the invention, the use ofnanoparticles having one or more different types of oligonucleotidesattached thereto is contemplated. The number of differentoligonucleotides attached to a nanoparticle and the lengths andsequences of the one or more oligonucleotides will contribute to therigidity and structural features of the resulting nanomaterials andnanostructures.

Also, the size, shape and chemical composition of the nanoparticles willcontribute to the properties of the resulting nanomaterials andnanostructures. These properties include optical properties,optoelectronic properties, electrochemical properties, electronicproperties, stability in various solutions, pore and channel sizevariation, ability to separate bioactive molecules while acting as afilter, etc. The use of mixtures of nanoparticles having differentsizes, shapes and/or chemical compositions, as well as the use ofnanoparticles having uniform sizes, shapes and chemical composition, arecontemplated.

In either fabrication method, the nanoparticles in the resultingnanomaterial or nanostructure are held together by oligonucleotideconnectors. The sequences, lengths, and strandedness of theoligonucleotide connectors, and the number of different oligonucleotideconnectors present will contribute to the rigidity and structuralproperties of the nanomaterial or nanostructure. If an oligonucleotideconnector is partially double-stranded, its rigidity can be increased bythe use of a filler oligonucleotide as described above in connectionwith the method of detecting nucleic acid. The rigidity of a completelydouble-stranded oligonucleotide connector can be increased by the use ofone or more reinforcing oligonucleotides having complementary sequencesso that they bind to the double-stranded oligonucleotide connector toform triple-stranded oligonucleotide connectors. The use ofquadruple-stranded oligonucleotide connectors based on deoxyquanosine ordeoxycytidine quartets is also contemplated.

Several of a variety of systems for organizing nanoparticles based onoligonucleotide hybridization are illustrated in the figures. In asimple system (FIG. 1) one set of nanoparticles bears oligonucleotideswith a defined sequence and another set of nanoparticles bearsoligonucleotides with a complementary sequence. On mixing the two setsof nanoparticle-oligonucleotide conjugates under hybridizationconditions, the two types of particles are linked by double strandedoligonucleotide connectors which serve as spacers to position thenanoparticles at selected distances.

An attractive system for spacing nanoparticles involves the addition ofone free linking oligonucleotide as illustrated in FIG. 2. The sequenceof the linking oligonucleotide will have at least a first portion and asecond portion for binding to oligonucleotides on nanoparticles. Thissystem is basically the same as utilized in the nucleic acid detectionmethod, except that the length of the added linking oligonucleotide canbe selected to be equal to the combined lengths of oligonucleotidesattached to the nanoparticles. The related system illustrated in FIG. 3provides a convenient means to tailor the distance between nanoparticleswithout having to change the sets of nanoparticle-oligonucleotideconjugates employed.

A further elaboration of the scheme for creating defined spaces betweennanoparticles is illustrated in FIG. 4. In this case a double strandedsegment of DNA or RNA containing overhanging ends is employed as thelinking oligonucleotide. Hybridization of the single-stranded,overhanging segments of the linking oligonucleotide with theoligonucleotides attached to the nanoparticles affords multipledouble-stranded oligonucleotide cross-links between the nanoparticles.

Stiffer nanomaterials and nanostructures, or portions thereof, can begenerated by employing triple-stranded oligonucleotide connectorsbetween nanoparticles. In forming the triple strand, one may exploiteither the pyrimidine:purine:pyrimidine motif (Moser, H. E. and Dervan,P. B. Science, 238,645–650 (1987) or the purine:purine:pyrimidine motif(Pilch, D. S. et al. Biochemistry, 30, 6081–6087 (1991). An example ofthe organization of nanoparticles by generating triple-strandedconnectors by the pyrimidine:purine:pyrimidine motif are illustrated inFIG. 10. In the system shown in FIG. 10, one set of nanoparticles isconjugated with a defined strand containing pyrimidine nucleotides andthe other set is conjugated with a complementary oligonucleotidecontaining purine nucleotides. Attachment of the oligonucleotides isdesigned such that the nanoparticles are separated by thedouble-stranded oligonucleotide formed on hybridization. Then, a freepyrimidine oligonucleotide with an orientation opposite that for thepyrimidine strand linked to the nanoparticle is added to the systemprior to, simultaneously with, or just subsequent to mixing thenanoparticles. Since the third strand in this system is held byHoogsteen base pairing, the triple strand is relatively unstablethermally. Covalent bridges spanning the breadth of the duplex are knownto stabilize triple-stranded complexes (Salunke, M., Wu, T., Letsinger,R. L., J. Am, Chem. Soc. 114, 8768–8772, (1992). Letsinger, R. L. andWu, T. J. Am Chem. Soc., 117, 7323–7328 (1995). Prakash, G. and Kool, J.Am. Chem. Soc., 114, 3523–3527 (1992).

For construction of nanomaterials and nanostructures, it may bedesirable in some cases to “lock” the assembly in place by covalentcross-links after formation of the nanomaterial or nanostructure byhybridization of the oligonucleotide components. This can beaccomplished by incorporating functional groups that undergo a triggeredirreversible reaction into the oligonucleotides. An example of afunctional group for this purpose is a stilbenedicarboxamide group. Ithas been demonstrated that two stilbenedicarboxamide groups alignedwithin hybridized oligonucleotides readily undergo cross-linking onirradiation with ultraviolet light (340 nm) (Lewis, F. D. et al. (1995)J Am. Chem. Soc. 117, 8785–8792).

Alternatively, one could employ the displacement of a 5′-O-tosyl groupfrom an oligonucleotide, held at the 3′-position to a nanoparticle by amercaptoalkly group, with a thiophosphoryl group at the 3′-end of anoligonucleotide held to an nanoparticle by a mercaptoalkyl group. In thepresence of an oligonucleotide that hybridizes to both oligonucleotidesand, thereby, brings the thiophosphoryl group into proximity of thetosyl group, the tosyl group will be displaced by the thiophosphorylgroup, generating an oligonucleotide linked at the ends to two differentnanoparticles. For displacement reactions of this type, see Herrlein etal., J. Am. Chem. Soc., 177, 10151–10152 (1995). The fact thatthiophosphoryl oligonucleotides do not react with gold nanoparticlesunder the conditions employed in attachingmercaptoalkyl-oligonucleotides to gold nanoparticles enables one toprepare gold nanoparticle-oligonucleotide conjugates anchored throughthe mercapto group to the nanoparticles and containing a terminalthiophosphoryl group free for the coupling reaction.

A related coupling reaction to lock the assembled nanoparticle system inplace utilizes displacement of bromide from a terminalbromoacetylaminonucleoside by a terminal thiophosphoryl-oligonucleotideas described in Gryaznov and Letsinger, J. Am. Chem. Soc., 115, 3808.This reaction proceeds much like the displacement of tosylate describedabove, except that the reaction is faster. Nanoparticles bearingoligonucleotides terminated with thiophosphoryl groups are prepared asdescribed above. For preparation of nanoparticles bearingoligonucleotides terminated with bromoacetylamino groups, one firstprepares an oligonucleotide terminated at one end by an aminonucleoside(e.g., either 5′-amino-5′-deoxythymidine or 3′-amino-3′-deoxythymidine)and at the other end by a mercaptoalkyl group. Molecules of thisoligonucleotide are then anchored to the nanoparticles through themercapto groups, and the nanoparticle-oligonucleotide conjugate is thenconverted the N-bromoacetylamino derivative by reaction with abromoacetyl acylating agent.

A fourth coupling scheme to lock the assemblies in place utilizesoxidation of nanoparticles bearing oligonucleotides terminated bythiophosphoryl groups. Mild oxidizing agents, such as potassiumtriiodide, potassium ferricyanide (see Gryaznov and Letsinger, NucleicAcids Research, 21, 1403) or oxygen, are preferred.

In addition, the properties of the nanomaterials and nanostructures canbe altered by incorporating into the interconnecting oligonucleotidechains organic and inorganic functions that are held in place bycovalent attachment to the oligonucleotide chains. A wide variety ofbackbone, base and sugar modifications are well known (see for exampleUhlmann, E., and Peyman, A. Chemical Reviews, 90, 544–584 (1990). Also,the oligonucleotide chains could be replaced by “Peptide Nucleic Acid”chains (PNA), in which the nucleotide bases are held by a polypeptidebackbone (see Wittung, P. et al., Nature, 368, 561–563 (1994).

As can be seen from the foregoing, the nanofabrication method of theinvention is extremely versatile. By varying the length, sequence andstrandedness of the linking oligonucleotides, the number, length, andsequence of the binding portions of the linking oligonucleotides, thelength, sequence and number of the oligonucleotides attached to thenanoparticles, the size, shape and chemical composition of thenanoparticles, the number and types of different linkingoligonucleotides and nanoparticles used, and the strandedness of theoligonucleotide connectors, nanomaterials and nanostructures having awide range of structures and properties can be prepared. Thesestructures and properties can be varied further by cross-linking of theoligonucleotide connectors, by functionalizing the oligonucleotides, bybackbone, base or sugar modifications of the oligonucleotides, or by theuse of peptide-nucleic acids.

The nanomaterials and nanostructures that can be made by thenanofabrication method of the invention include nanoscale mechanicaldevices, separation membranes, bio-filters, and biochips. It iscontemplated that the nanomaterials and nanostructures of the inventioncan be used as chemical sensors, in computers, for drug delivery, forprotein engineering, and as templates for biosynthesis/nanostructurefabrication/directed assembly of other structures. See generally Seemanet al., New J. Chem., 17, 739 (1993) for other possible applications.The nanomaterials and nanostructures that can be made by thenanofabrication method of the invention also can include electronicdevices. Whether nucleic acids could transport electrons has been thesubject of substantial controversy. As shown in Example 21 below,nanoparticles assembled by DNA conduct electricity (the DNA connectorsfunction as semiconductors).

Finally, the invention provides methods of making uniquenanoparticle-oligonucleotide conjugates. In the first such method,oligonucleotides are bound to charged nanoparticles to produce stablenanoparticle-oligonucleotide conjugates. Charged nanoparticles includenanoparticles made of metal, such as gold nanoparticles.

The method comprises providing oligonucleotides having covalently boundthereto a moiety comprising a functional group which can bind to thenanoparticles. The moieties and functional groups are those describedabove for binding (i.e., by chemisorption or covalent bonding)oligonucleotides to nanoparticles. For instance, oligonucleotides havingan alkanethiol or an alkanedisulfide covalently bound to their 5′ or 3′ends can be used to bind the oligonucleotides to a variety ofnanoparticles, including gold nanoparticles.

The oligonucleotides are contacted with the nanoparticles in water for atime sufficient to allow at least some of the oligonucleotides to bindto the nanoparticles by means of the functional groups. Such times canbe determined empirically. For instance, it has been found that a timeof about 12–24 hours gives good results. Other suitable conditions forbinding of the oligonucleotides can also be determined empirically. Forinstance, a concentration of about 10–20 nM nanoparticles and incubationat room temperature gives good results.

Next, at least one salt is added to the water to form a salt solution.The salt can be any water-soluble salt. For instance, the salt may besodium chloride, magnesium chloride, potassium chloride, ammoniumchloride, sodium acetate, ammonium acetate, a combination of two or moreof these salts, or one of these salts in phosphate buffer. Preferably,the salt is added as a concentrated solution, but it could be added as asolid. The salt can be added to the water all at one time or the salt isadded gradually over time. By “gradually over time” is meant that thesalt is added in at least two portions at intervals spaced apart by aperiod of time. Suitable time intervals can be determined empirically.

The ionic strength of the salt solution must be sufficient to overcomeat least partially the electrostatic repulsion of the oligonucleotidesfrom each other and, either the electrostatic attraction of thenegatively-charged oligonucleotides for positively-chargednanoparticles, or the electrostatic repulsion of the negatively-chargedoligonucleotides from negatively-charged nanoparticles. Graduallyreducing the electrostatic attraction and repulsion by adding the saltgradually over time has been found to give the highest surface densityof oligonucleotides on the nanoparticles. Suitable ionic strengths canbe determined empirically for each salt or combination of salts. A finalconcentration of sodium chloride of from about 0.1 M to about 1.0 M inphosphate buffer, preferably with the concentration of sodium chloridebeing increased gradually over time, has been found to give goodresults.

After adding the salt, the oligonucleotides and nanoparticles areincubated in the salt solution for an additional period of timesufficient to allow sufficient additional oligonucleotides to bind tothe nanoparticles to produce the stable nanoparticle-oligonucleotideconjugates. As will be described in detail below, an increased surfacedensity of the oligonucleotides on the nanoparticles has been found tostabilize the conjugates. The time of this incubation can be determinedempirically. A total incubation time of about 24–48, preferably 40hours, has been found to give good results (this is the total time ofincubation; as noted above, the salt concentration can be increasedgradually over this total time). This second period of incubation in thesalt solution is referred to herein as the “aging” step. Other suitableconditions for this “aging” step can also be determined empirically. Forinstance, incubation at room temperature and pH 7.0 gives good results.

The conjugates produced by use of the “aging” step have been found to beconsiderably more stable than those produced without the “aging” step.As noted above, this increased stability is due to the increased densityof the oligonucleotides on the surfaces of the nanoparticles which isachieved by the “aging” step. The surface density achieved by the“aging” step will depend on the size and type of nanoparticles and onthe length, sequence and concentration of the oligonucleotides. Asurface density adequate to make the nanoparticles stable and theconditions necessary to obtain it for a desired combination ofnanoparticles and oligonucleotides can be determined empirically.Generally, a surface density of at least 10 picomoles/cm² will beadequate to provide stable nanoparticle-oligonucleotide conjugates.Preferably, the surface density is at least 15 picomoles/cm². Since theability of the oligonucleotides of the conjugates to hybridize withnucleic acid and oligonucleotide targets can be diminished if thesurface density is too great, the surface density is preferably nogreater than about 35–40 picomoles/cm².

As used herein, “stable” means that, for a period of at least six monthsafter the conjugates are made, a majority of the oligonucleotides remainattached to the nanoparticles and the oligonucleotides are able tohybridize with nucleic acid and oligonucleotide targets under standardconditions encountered in methods of detecting nucleic acid and methodsof nanofabrication.

Aside from their stability, the nanoparticle-oligonucleotide conjugatesmade by this method exhibit other remarkable properties. See, e.g.,Examples 5, 7, and 19 of the present application. In particular, due tothe high surface density of the conjugates, they will assemble intolarge aggregates in the presence of a target nucleic acid oroligonucleotide. The temperature over which the aggregates form anddissociate has unexpectedly been found to be quite narrow, and thisunique feature has important practical consequences. In particular, itincreases the selectivity and sensitivity of the methods of detection ofthe present invention. A single base mismatch and as little as 20femtomoles of target can be detected using the conjugates. Althoughthese features were originally discovered in assays performed insolution, the advantages of the use of these conjugates have been foundto extend to assays performed on substrates, including those in whichonly a single type of conjugate is used.

It has been found that the hybridization efficiency ofnanoparticle-oligonucleotide conjugates can be increased dramatically bythe use of recognition oligonucleotides which comprise a recognitionportion and a spacer portion. “Recognition oligonucleotides” areoligonucleotides which comprise a sequence complementary to at least aportion of the sequence of a nucleic acid or oligonucleotide target. Inthis embodiment, the recognition oligonucleotides comprise a recognitionportion and a spacer portion, and it is the recognition portion whichhybridizes to the nucleic acid or oligonucleotide target. The spacerportion of the recognition oligonucleotide is designed so that it canbind to the nanoparticles. For instance, the spacer portion could have amoiety covalently bound to it, the moiety comprising a functional groupwhich can bind to the nanoparticles. These are the same moieties andfunctional groups as described above. As a result of the binding of thespacer portion of the recognition oligonucleotide to the nanoparticles,the recognition portion is spaced away from the surface of thenanoparticles and is more accessible for hybridization with its target.The length and sequence of the spacer portion providing good spacing ofthe recognition portion away from the nanoparticles can be determinedempirically. It has been found that a spacer portion comprising at leastabout 10 nucleotides, preferably 10–30 nucleotides, gives good results.The spacer portion may have any sequence which does not interfere withthe ability of the recognition oligonucleotides to become bound to thenanoparticles or to a nucleic acid or oligonucleotide target. Forinstance, the spacer portions should not sequences complementary to eachother, to that of the recognition olignucleotides, or to that of thenucleic acid or oligonucleotide target of the recognitionoligonucleotides. Preferably, the bases of the nucleotides of the spacerportion are all adenines, all thymines, all cytidines, or all guanines,unless this would cause one of the problems just mentioned. Morepreferably, the bases are all adenines or all thymines. Most preferablythe bases are all thymines.

It has further been found that the use of diluent oligonucleotides inaddition to recognition oligonucleotides provides a means of tailoringthe conjugates to give a desired level of hybridization. The diluent andrecognition oligonucleotides have been found to attach to thenanoparticles in about the same proportion as their ratio in thesolution contacted with the nanoparticles to prepare the conjugates.Thus, the ratio of the diluent to recognition oligonucleotides bound tothe nanoparticles can be controlled so that the conjugates willparticipate in a desired number of hybridization events. The diluentoligonucleotides may have any sequence which does not interfere with theability of the recognition oligonucleotides to be bound to thenanoparticles or to bind to a nucleic acid or oligonucleotide target.For instance, the diluent oligonulceotides should not have a sequencecomplementary to that of the recognition olignucleotides or to that ofthe nucleic acid or oligonucleotide target of the recognitionoligonucleotides. The diluent oligonucleotides are also preferably of alength shorter than that of the recognition oligonucleotides so that therecognition oligonucleotides can bind to their nucleic acid oroligonucleotide targets. If the recognition oligonucleotides comprisespacer portions, the diluent oligonulceotides are, most preferably,about the same length as the spacer portions. In this manner, thediluent oligonucleotides do not interefere with the ability of therecognition portions of the recognition oligonucleotides to hybridizewith nucleic acid or oligonucleotide targets. Even more preferably, thediluent oligonucleotides have the same sequence as the sequence of thespacer portions of the recognition oligonucleotides.

As can be readily appreciated, highly desirablenanoparticle-oligonucleotide conjugates can be prepared by employing allof the methods described above. By doing so, stable conjugates withtailored hybridization abilities can be produced.

Any of the above conjugates can be, and are preferably, used in any ofthe methods of detecting nucleic acids described above, and theinvention also provides a kit comprising a container holding any of theabove conjugates. In addition, the conjugates can be, and arepreferably, used in any of the methods of nanofabrication of theinvention and the method of separating nucleic acids.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity. For example, “a characteristic” refers to one or morecharacteristics or at least one characteristic. As such, the terms “a”(or “an”), “one or more” and “at least one” are used interchangeablyherein. It is also to be noted that the terms “comprising”, “including”,and “having” have been used interchangeably.

EXAMPLES Example 1 Preparation of Oligonucleotide-Modified GoldNanoparticles A. Preparation of Gold Nanoparticles

Gold colloids (13 nm diameter) were prepared by reduction of HAuCl₄ withcitrate as described in Frens, Nature Phys. Sci., 241, 20 (1973) andGrabar, Anal. Chem., 67, 735 (1995). Briefly, all glassware was cleanedin aqua regia (3 parts HCl, 1 part HNO₃), rinsed with Nanopure H₂O, thenoven dried prior to use. HAuCl₄ and sodium citrate were purchased fromAldrich Chemical Company. Aqueous HAuCl₄ (1 mM, 500 mL) was brought toreflux while stirring. Then, 38.8 mM sodium citrate (50 mL) was addedquickly. The solution color changed from pale yellow to burgundy, andrefluxing was continued for 15 min. After cooling to room temperature,the red solution was filtered through a Micron Separations Inc. 1 micronfilter. Au colloids were characterized by UV-vis spectroscopy using aHewlett Packard 8452A diode array spectrophotometer and by TransmissionElectron Microscopy (TEM) using a Hitachi 8100 transmission electronmicroscope. Gold particles with diameters of 13 nm will produce avisible color change when aggregated with target and probeoligonucleotide sequences in the 10–35 nucleotide range.

B. Synthesis Of Oligonucleotides

Oligonucleotides were synthesized on a 1 micromole scale using aMilligene Expedite DNA synthesizer in single column mode usingphosphoramidite chemistry. Eckstein, F. (ed.) Oligonucleotides andAnalogues: A Practical Approach (IRL Press, Oxford, 1991). All solutionswere purchased from Milligene (DNA synthesis grade). Average couplingefficiency varied from 98 to 99.8%, and the final dimethoxytrityl (DMT)protecting group was not cleaved from the oligonucleotides to aid inpurification.

For 3′-thiol-oligonucleotides, Thiol-Modifier C3 S—S CPG support waspurchased from Glen Research and used in the automated synthesizer.During normal cleavage from the solid support (16 hr at 55° C.), 0.05 Mdithiothreitol (DTT) was added to the NH₄OH solution to reduce the 3′disulfide to the thiol. Before purification by reverse phase highpressure liquid chromatography (HPLC), excess DTT was removed byextraction with ethyl acetate.

For 5′-thiol oligonucleotides, 5′-Thiol-Modifier C₆-phosphoramiditereagent was purchased from Glen Research, 44901 Falcon Place, Sterling,Va. 20166. The oligonucleotides were synthesized, and the final DMTprotecting group removed. Then, 1 ml of dry acetonitrile was added to100 μmole of the 5′ Thiol Modifier C₆-phosphoramidite. 200 μL of theamidite solution and 200 μL of activator (fresh from synthesizer) weremixed and introduced onto the column containing the synthesizedoligonucleotides still on the solid support by syringe and pumped backand forth through the column for 10 minutes. The support was then washed(2×1 mL) with dry acetonitrile for 30 seconds. 700 μL of a 0.016 MI₂/H₂O/pyridine mixture (oxidizer solution) was introduced into thecolumn, and was then pumped back and forth through the column with twosyringes for 30 second. The support was then washed with a 1:1 mixtureof CH₃CN/pyridine (2×1 mL) for 1 minute, followed by a final wash withdry acetonitrile (2×1 mL) with subsequent drying of the column with astream of nitrogen. The trityl protecting group was not removed, whichaids in purification.

Reverse phase HPLC was performed with a Dionex DX500 system equippedwith a Hewlett Packard ODS hypersil column (4.6×200 mm, 5 mm particlesize) using 0.03 M Et₃NH⁺ OAc⁻ buffer (TEAA), pH 7, with a 1%/min.gradient of 95% CH₃CN/5% TEAA. The flow rate was 1 mL/min. with UVdetection at 260 nm. Preparative HPLC was used to purify theDMT-protected unmodified oligonucleotides (elution at 27 min). Aftercollection and evaporation of the buffer, the DMT was cleaved from theoligonucleotides by treatment with 80% acetic acid for 30 min at roomtemperature. The solution was then evaporated to near dryness, water wasadded, and the cleaved DMT was extracted from the aqueousoligonucleotide solution using ethyl acetate. The amount ofoligonucleotide was determined by absorbance at 260 nm, and final purityassessed by reverse phase HPLC (elution time 14.5 minutes).

The same protocol was used for purification of the3′-thiol-oligonucleotides, except that DTT was added after extraction ofDMT to reduce the amount of disulfide formed.

After six hours at 40° C., the DTT was extracted using ethyl acetate,and the oligonucleotides repurified by HPLC (elution time 15 minutes).

For purification of the 5′ thiol modified oligonucleotides, preparatoryHPLC was performed under the same conditions as for unmodifiedoligonucleotides. After purification, the trityl protecting group wasremoved by adding 150 μL of a 50 mM AgNO₃ solution to the dryoligonucleotide sample. The sample turned a milky white color as thecleavage occurred. After 20 minutes, 200 μL of a 10 mg/ml solution ofDTT was added to complex the Ag (five minute reaction time), and thesample was centrifuged to precipitate the yellow complex. Theoligonucleotide solution (<50 OD) was then transferred onto a desaltingNAP-5 column (Pharmacia Biotech, Uppsala, Sweden) for purification(contains DNA Grade Sephadex G-25 Medium for desalting and bufferexchange of oligonucleotides greater than 10 bases). The amount of 5′thiol modified oligonucleotide was determined by UV-vis spectroscopy bymeasuring the magnitude of the absorbance at 260 nm. The final puritywas assessed by performing ion-exchange HPLC with a Dionex NucleopacPA-100 (4×250) column using a 10 mM NaOH solution (pH 12) with a 2%/mingradient of 10 mM NaOH, 1M NaCl solution. Typically, two peaks resultedwith elution times of approximately 19 minutes and 25 minutes (elutiontimes are dependent on the length of the oligonucleotide strand). Thesepeaks corresponded to the thiol and the disulfide oligonucleotidesrespectively.

C. Attachment of Oligonucleotides to Gold Nanoparticles

An aqueous solution of 17 nM (150 μL) Au colloids, prepared as describedin part A above, was mixed with 3.75 μM (46 μL) 3′-thiol-TTTGCTGA,prepared as described in part B and allowed to stand for 24 hours atroom temperature in 1 ml Eppendorf capped vials. A second solution ofcolloids was reacted with 3.75 μM (46 μL) 3′-thiol-TACCGTTG. Note thatthese oligonucleotides are noncomplementary. Shortly before use, equalamounts of each of the two nanoparticle solutions were combined. Sincethe oligonucleotides are noncomplementary, no reaction took place.

The oligonucleotide-modified nanoparticles are stable at elevatedtemperatures (80° C.) and high salt concentrations (1M NaCl) for daysand have not been observed to undergo particle growth. Stability in highsalt concentrations is important, since such conditions are required forthe hybridization reactions that form the basis of the methods ofdetection and nanofabrication of the invention.

Example 2 Formation of Nanoparticle Aggregates A. Preparation of LinkingOligonucleotide

Two (nonthiolated) oligonucleotides were synthesized as described inpart B of Example 1. They had the following sequences:

3′ ATATGCGCGA TCTCAGCAAA [SEQ ID NO:1]; and 3′ GATCGCGCAT ATCAACGGTA[SEQ ID NO:2].

Mixing of these two oligonucleotides in a 1 M NaCl, 10 mM phosphatebuffered (pH 7.0) solution, resulted in hybridization to form a duplexhaving a 12-base-pair overlap and two 8-base-pair sticky ends. Each ofthe sticky ends had a sequence which was complementary to that of one ofthe oligonucleotides attached to the Au colloids prepared in part C ofExample 1.

B. Formation of Nanoparticle Aggregates

The linking oligonucleotides prepared in part A of this example (0.17 μMfinal concentration after dilution with NaCl) were added to thenanoparticle-oligonucleotide conjugates prepared in part C of Example 1(5.1 nM final concentration after dilution with NaCl) at roomtemperature. The solution was then diluted with aqueous NaCl (to a finalconcentration of 1 M) and buffered at pH 7 with 10 mM phosphate,conditions which are suitable for hybridization of the oligonucleotides.An immediate color change from red to purple was observed, and aprecipitation reaction ensued. See FIG. 6. Over the course of severalhours, the solution became clear and a pinkish-gray precipitate settledto the bottom of the reaction vessel. See FIG. 6.

To verify that this process involved both the oligonucleotides andcolloids, the precipitate was collected and resuspended (by shaking) in1 M aqueous NaCl buffered at pH 7. Any of the oligonucleotides nothybridized to the nanoparticles are removed in this manner. Then, atemperature/time dissociation experiment was performed by monitoring thecharacteristic absorbance for the hybridized oligodeoxyribonucleotides(260 nm) and for the aggregated colloids which is reflective of the goldinterparticle distance (700 nm). See FIG. 7.

Changes in absorbance at 260 and 700 nm were recorded on a Perkin-ElmerLambda 2 UV-vis Spectrophotometer using a Peltier PTP-1 TemperatureControlled Cell Holder while cycling the temperature at a rate of 1°C./minute between 0° C. and 80° C. DNA solutions were approximately 1absorbance unit(s) (OD), buffered at pH 7 using 10 mM phosphate bufferand at 1M NaCl concentration.

The results are shown in FIG. 8A. As the temperature was cycled between0° C. and 80° C. (which is 38° C. above the dissociation temperature(T_(m)) for the duplex (T_(m)=42° C.)), there was an excellentcorrelation between the optical signatures for both the colloids andoligonucleotides. The UV-vis spectrum for naked Au colloids was muchless temperature dependent, FIG. 8B.

There was a substantial visible optical change when the polymericoligonucleotide-colloid precipitate was heated above its melting point.The clear solution turned dark red as the polymeric biomaterialde-hybridized to generate the unlinked colloids which are soluble in theaqueous solution. The process was reversible, as evidenced by thetemperature traces in FIG. 8A.

In a control experiment, a 14-T:14-A duplex was shown to be ineffectiveat inducing reversible Au colloid particle aggregation. In anothercontrol experiment, a linking oligonucleotide duplex with four base pairmismatches in the sticky ends was found not to induce reversibleparticle aggregation of oligonucleotide-modified nanoparticles (preparedas described in part C of Example 1 and reacted as described above). Ina third control experiment, non-thiolated oligonucleotides havingsequences complementary to the sticky ends of the linkingoligonucleotide and reacted with nanoparticles did not producereversible aggregation when the nanoparticles were combined with thelinking oligonucleotide.

Further evidence of the polymerization/assembly process came fromTransmission Electron Microscopy (TEM) studies of the precipitate. TEMwas performed on a Hitachi 8100 Transmission Electron Microscope. Atypical sample was prepared by dropping 100 μL of colloid solution ontoa holey carbon grid. The grid, then, was dried under vacuum and imaged.TEM images of Au colloids linked by hybridized oligonucleotides showedlarge assembled networks of the Au colloids, FIG. 9A. Naked Au colloidsdo not aggregate under comparable conditions but rather disperse orundergo particle growth reactions. Hayat, Colloidal Gold: Principles,Methods, and Applications (Academic Press, San Diego, 1991). Note thatthere is no evidence of colloid particle growth in the experimentsperformed to date; the hybridized colloids seem to be remarkably regularin size with an average diameter of 13 nm.

With TEM, a superposition of layers is obtained, making it difficult toassess the degree of order for three-dimensional aggregates. However,smaller scale images of single layer, two-dimensional aggregatesprovided more evidence for the self-assembly process, FIG. 9B.Close-packed assemblies of the aggregates with uniform particleseparations of approximately 60 Å can be seen. This distance is somewhatshorter than the estimated 95 Å spacing expected for colloids connectedby rigid oligonucleotide hybrids with the sequences that were used.However, because of the nicks in the duplex obtained after hybridizationof the oligonucleotides on the nanoparticles to the linkingoligonucleotides, these were not rigid hybrids and were quite flexible.It should be noted that this is a variable that can be controlled byreducing the system from four overlapping strands to three (therebyreducing the number of nicks) or by using triplexes instead of duplexes.

Example 3 Preparation of Oligonucleotide-Modified Gold Nanoparticles

Gold colloids (13 nm diameter) were prepared as described in Example 1.Thiol-oligonucleotides [HS(CH₂)₆OP(O)(O⁻)-oligonucleotide] were alsoprepared as described in Example 1.

The method of attaching thiol-oligonucleotides to gold nanoparticlesdescribed in Example 1 was found not to produce satisfactory results insome cases. In particular, when long oligonucleotides were used, theoligonucleotide-colloid conjugates were not stable in the presence of alarge excess of high molecular weight salmon sperm DNA used as model forthe background DNA that would normally be present in a diagnosticsystem. Longer exposure of the colloids to the thiol-oligonucleotidesproduced oligonucleotide-colloid conjugates that were stable to salmonsperm DNA, but the resulting conjugates failed to hybridizesatisfactorily. Further experimentation led to the following procedurefor attaching thiol-oligonucleotides of any length to gold colloids sothat the conjugates are stable to high molecular weight DNA andhybridize satisfactorily.

A 1 mL solution of the gold colloids (17 nM) in water was mixed withexcess (3.68 μM) thiol-oligonucleotide (28 bases in length) in water,and the mixture was allowed to stand for 12–24 hours at roomtemperature. Then, 100 μL of a 0.1 M sodium hydrogen phosphate buffer,pH 7.0, and 100 μL of 1.0 M NaCl were premixed and added. After 10minutes, 10 μL of 1% aqueous NaN₃ were added, and the mixture wasallowed to stand for an additional 40 hours. This “aging” step wasdesigned to increase the surface coverage by the thiol-oligonucleotidesand to displace oligonucleotide bases from the gold surface. Somewhatcleaner, better defined red spots in subsequent assays were obtained ifthe solution was frozen in a dry-ice bath after the 40-hour incubationand then thawed at room temperature. Either way, the solution was nextcentrifuged at 14,000 rpm in an Eppendorf Centrifuge 5414 for about 15minutes to give a very pale pink supernatant containing most of theoligonucleotide (as indicated by the absorbance at 260 nm) along with7–10% of the colloidal gold (as indicated by the absorbance at 520 nm),and a compact, dark, gelatinous residue at the bottom of the tube. Thesupernatant was removed, and the residue was resuspended in about 200 μLof buffer (10 mM phosphate, 0.1 M NaCl) and recentrifuged. After removalof the supernatant solution, the residue was taken up in 1.0 mL ofbuffer (10 mM phosphate, 0.1 M NaCl) and 10 μL of a 1% aqueous solutionof NaN₃. Dissolution was assisted by drawing the solution into, andexpelling it from, a pipette several times. The resulting red mastersolution was stable (i.e., remained red and did not aggregate) onstanding for months at room temperature, on spotting on silicathin-layer chromatography (TLC) plates (see Example 4), and on additionto 2 M NaCl, 10 mM MgCl₂, or solutions containing high concentrations ofsalmon sperm DNA.

Example 4 Acceleration Of Hybridization of Nanoparticle-OligonucleotideConjugates

The oligonucleotide-gold colloid conjugates I and II illustrated in FIG.11 were prepared as described in Example 3. The hybridization of thesetwo conjugates was extremely slow. In particular, mixing samples ofconjugates I and II in aqueous 0.1 M NaCl or in 10 mM MgCl₂ plus 0.1 MNaCl and allowing the mixture to stand at room temperature for a dayproduced little or no color change.

Two ways were found to improve hybridization. First, faster results wereobtained by freezing the mixture of conjugates I and II (each 15 nMcontained in a solution of 0.1 M NaCl) in a dry ice-isopropyl alcoholbath for 5 minutes and then thawing the mixture at room temperature. Thethawed solution exhibited a bluish color. When 1 μL of the solution wasspotted on a standard C-18 TLC silica plate (Alltech Associates), astrong blue color was seen immediately. The hybridization and consequentcolor change caused by the freeze-thawing procedure were reversible. Onheating the hybridized solution to 80° C., the solution turned red andproduced a pink spot on a TLC plate. Subsequent freezing and thawingreturned the system to the (blue) hybridized state (both solution andspot on a C-18 TLC plate). In a similar experiment in which the solutionwas not refrozen, the spot obtained on the C-18 TLC plate was pink.

A second way to obtain faster results is to warm the conjugates andtarget. For instance, in another experiment, oligonucleotide-goldcolloid conjugates and an oligonucleotide target sequence in a 0.1 MNaCl solution were warmed rapidly to 65° C. and allowed to cool to roomtemperature over a period of 20 minutes. On spotting on a C-18 silicaplate and drying, a blue spot indicative of hybridization was obtained.In contrast, incubation of the conjugates and target at room temperaturefor an hour in 0.1 M NaCl solution did not produce a blue colorindicative of hybridization. Hybridization is more rapid in 0.3 M NaCl.

Example 5 Assays Using Nanoparticle-Oligonucleotide Conjugates

The oligonucleotide-gold colloid conjugates 1 and 2 illustrated in FIGS.12A–F were prepared as described in Example 3, and the oligonucleotidetarget 3 illustrated in FIG. 12A was prepared as described in Example 2.Mismatched and deletion targets 4, 5, 6, and 7 were purchased from theNorthwestern University Biotechnology Facility, Chicago, Ill. Theseoligonucleotides were synthesized on a 40 nmol scale and purified on anreverse phase C18 cartridge (OPC). Their purity was determined byperforming ion exchange HPLC.

Selective hybridization was achieved by heating rapidly and then coolingrapidly to the stringent temperature. For example, hybridization wascarried out in 100 μL of 0.1 M NaCl plus 5 mM MgCl₂ containing 15 nM ofeach oligonucleotide-colloid conjugate 1 and 2, and 3 nanomoles oftarget oligonucleotide 3, 4, 5, 6, or 7, heating to 74° C., cooling tothe temperatures indicated in Table 1 below, and incubating the mixtureat this temperature for 10 minutes. A 3 μL sample of each reactionmixture was then spotted on a C-18 TLC silica plate. On drying (5minutes), a strong blue color appeared if hybridization had taken place.

The results are presented in Table 1 below. Pink spots signify anegative test (i.e., that the nanoparticles were not brought together byhybridization), and blue spots signify a positive test (i.e., that thenanoparticles were brought into proximity due to hybridization involvingboth of the oligonucleotide-colloid conjugates).

TABLE 1 Results (Color) Reactants 45° C. 50° C. 60° C. 74° C. 1 + 2 PinkPink Pink Pink 1 + 2 + 3 (match) Blue Blue Blue Blue 1 + 2 + 4 (halfcomplement Pink Pink Pink Pink mismatch) 1 + 2 + 5 (−6 bp) Blue PinkPink Pink 1 + 2 + 6 (1 bp mismatch) Blue Blue Pink Pink 1 + 2 + 7 (2 bpmismatch) Pink Pink Pink Pink

As can be seen in Table 1, hybridization at 60° C. gave a blue spot onlyfor the fully-matched target 3. Hybridization at 50° C. yielded bluespots with both targets 3 and 6. Hybridization at 45° C. gave blue spotswith targets 3, 5 and 6.

In a related series, a target containing a single mismatch T nucleotidewas found to give a positive test at 58° C. (blue color) and a negativetest (red color) at 64° C. with conjugates 1 and 2. Under the sameconditions, the fully-matched target (3) gave a positive test at bothtemperatures, showing that the test can discriminate between a targetthat is fully matched and one containing a single mismatched base.

Similar results were achieved using a different hybridization method. Inparticular, selective hybridization was achieved by freezing, thawingand then warming rapidly to the stringent temperature. For example,hybridization was carried out in 100 μL of 0.1 M NaCl containing 15 nMof each oligonucleotide-colloid conjugate 1 and 2, and 10 picomoles oftarget oligonucleotide 3, 4, 5, 6, or 7, freezing in a dry ice-isopropylalcohol bath for 5 minutes, thawing at room temperature, then warmingrapidly to the temperatures indicated in Table 2 below, and incubatingthe mixture at this temperature for 10 minutes. A 3 μL sample of eachreaction mixture was then spotted on a C-18 TLC silica plate. Theresults are presented in Table 2.

TABLE 2 Results Reactants (probes) + (color) target RT 35° C. 40° C. 54°C. 64° C. (1 + 2) + 3 blue blue blue blue pink (1 + 2) pink pink pinkpink pink (1 + 2) + 4 pink pink pink pink pink (1 + 2) + 5 blue bluepink pink pink (1 + 2) + 6 blue blue blue pink pink (1 + 2) + 7 bluepink pink pink pink

An important feature of these systems was that the color changeassociated with the temperature change was very sharp, occurring over atemperature range of about 1° C. This indicates high cooperativity inthe melting and association processes involving the colloid conjugatesand enables one to easily discriminate between oligonucleotide targetscontaining a fully-matched sequence and a single basepair mismatch.

The high degree of discrimination may be attributed to two features. Thefirst is the alignment of two relatively short probe oligonucleotidesegments (15 nucleotides) on the target is required for a positivesignal. A mismatch in either segment is more destabilizing than amismatch in a longer probe (e.g., an oligonucleotide 30 bases long) in acomparable two-component detection system. Second, the signal at 260 nm,obtained on hybridization of the target oligonucleotides with thenanoparticle conjugates in solution, is nanoparticle-based, notDNA-based. It depends on dissociation of an assembly of nanoparticlesorganized in a polymeric network by multiple oligonucleotide duplexes.This results in a narrowing of the temperature range that is observedfor aggregate dissociation, as compared with standard DNA thermaldenaturation. In short, some duplexes in the crosslinked aggregates candissociate without dispersing the nanoparticles into solution.Therefore, the temperature range for aggregate melting is very narrow(4° C.) as compared with the temperature range associated with meltingthe comparable system without nanoparticles (12° C.). Even more strikingand advantageous for this detection approach, is the temperature rangefor the colorimetric response (<1° C.) observe on the C18 silica plates.In principle, this three-component nanoparticle based strategy will bemore selective than any two-component detection system based on asingle-strand probe hybridizing with target nucleic acid.

A master solution containing 1 nmol of target 3 was prepared in 100 μlof hybridization buffer (0.3 M NaCl, 10 mM phosphate, pH 7). One μl ofthis solution corresponds to 10 picomole of target oligonucleotide.Serial dilutions were performed by taking an aliquot of the mastersolution and diluting it to the desired concentration with hybridizationbuffer. Table 3 shows the sensitivity obtained using 3 μl of a mixtureof probes 1 and 2 with different amounts of target 3. After performingthe hybridization using freeze-thaw conditions, 3 μl aliquots of thesesolutions were spotted onto C-18 TLC plates to determine color. In Table3 below, pink signifies a negative test, and blue signifies a positivetest.

TABLE 3 Amount of Target Results 1 picomole blue (positive) 200femtomole blue (positive) 100 femtomole blue (positive) 20 femtomoleblue (positive) 10 femtomole purplish (ambiguous)This experiment indicates that 10 femtomoles is the lower limit ofdetection for this particular system.

Example 6 Assays Using Nanoparticle-Oligonucleotide Conjugates

DNA modified nanoparticles were adsorbed onto modified transparentsubstrates as shown in FIG. 13B. This method involved the linking of DNAmodified nanoparticles to nanoparticles that were attached to a glasssubstrate, using DNA hybridization interactions.

Glass microscope slides were purchased from Fisher scientific. Slideswere cut into approximately 5×15 mm pieces, using a diamond tippedscribing pen. Slides were cleaned by soaking for 20 minutes in asolution of 4:1H₂SO₄:H₂O₂ at 50° C. Slides were then rinsed with copiousamounts of water, then ethanol, and dried under a stream of drynitrogen. To functionalize the slide surface with a thiol terminatedsilane, the slides were soaked in a degassed ethanolic 1% (by volume)mercaptopropyl-trimethoxysilane solution for 12 hours. The slides wereremoved from the ethanol solutions and rinsed with ethanol, then water.Nanoparticles were adsorbed onto the thiol terminated surface of theslides by soaking in solutions containing the 13 nm diameter goldnanoparticles (preparation described in Example 1). After 12 hours inthe colloidal solutions, the slides were removed and rinsed with water.The resulting slides have a pink appearance due to the adsorbednanoparticles and exhibit similar UV-vis absorbance profiles (surfaceplasmon absorbance peak at 520 nm) as the aqueous gold nanoparticlecolloidal solutions. See FIG. 14A.

DNA was attached to the nanoparticle modified surface by soaking theglass slides in 0.2 OD (1.7 μM) solution containing freshly purified 3′thiol oligonucleotide (3′ thiol ATGCTCAACTCT [SEQ ID NO:33])(synthesized as described in Examples 1 and 3). After 12 hours ofsoaking time, the slides were removed and rinsed with water.

To demonstrate the ability of an analyte DNA strand to bindnanoparticles to the modified substrate, a linking oligonucleotide wasprepared. The linking oligonucleotide (prepared as described in Example2) was 24 bp long (5′ TACGAGTTGAGAATCCTGAATGCG [SEQ ID NO:34]) with asequence containing a 12 bp end that was complementary to the DNAalready adsorbed onto the substrate surface (SEQ ID NO:33). Thesubstrate was then soaked in a hybridization buffer (0.5 M NaCl, 10 mMphosphate buffer pH 7) solution containing the linking oligonucleotide(0.4 OD, 1.7 μM) for 12 hours. After removal and rinsing with similarbuffer, the substrate was soaked in a solution containing 13 nm diametergold nanoparticles which had been modified with an oligonucleotide(TAGGACTTACGC 5′ thiol [SEQ ID NO:35]) (prepared as described in Example3) that is complementary to the unhybridized portion of the linkingoligonucleotide attached to the substrate. After 12 hours of soaking,the substrate was removed and rinsed with the hybridization buffer. Thesubstrate color had darkened to a purple color and the UV-vis absorbanceat 520 nm approximately doubled (FIG. 14A).

To verify that the oligonucleotide modified gold nanoparticles wereattached to the oligonucleotide/nanoparticle modified surface throughDNA hybridization interactions with the linking oligonucleotide, amelting curve was performed. For the melting experiment, the substratewas placed in a cuvette containing 1 mL of hybridization buffer and thesame apparatus used in Example 2, part B, was used. The absorbancesignal due to the nanoparticles (520 nm) was monitored as thetemperature of the substrate was increased at a rate of 0.5° C. perminute. The nanoparticle signal dramatically dropped when thetemperature passed 60° C. See FIG. 14B. A first derivative of the signalshowed a melting temperature of 62° C., which corresponds with thetemperature seen for the three DNA sequences hybridized in solutionwithout nanoparticles. See FIG. 14B.

Example 7 Assays Using Nanoparticle-Oligonucleotide Conjugates

The detection system illustrated in FIGS. 15A–G was designed so that thetwo probes 1 and 2 align in a tail-to-tail fashion onto a complementarytarget 4 (see FIGS. 15A–G). This differs from the system described inExample 5 where the two probes align contiguously on the target strand(see FIGS. 12A–F).

The oligonucleotide-gold nanoparticle conjugates 1 and 2 illustrated inFIGS. 15A–G were prepared as described in Example 3, except that thenanoparticles were redispersed in hybridization buffer (0.3 M NaCl, 10mM phosphate, pH 7). The final nanoparticle-oligonucleotide conjugateconcentration was estimated to be 13 nM by measuring the reduction inintensity of the surface plasmon band at 522 nm which gives rise to thered color of the nanoparticles. The oligonucleotide targets illustratedin FIGS. 15A–G were purchased from the Northwestern UniversityBiotechnology Facility, Evanston, Ill.

When 150 μL of hybridization buffer containing 13 nMoligonucleotide-nanoparticle conjugates 1 and 2 was mixed with 60picomoles (6 μL) of target 4, the solution color immediately changedfrom red to purple. This color change occurs as a result of theformation of large oligonucleotide-linked polymeric networks of goldnanoparticles, which leads to a red shift in the surface plasmonresonance of the nanoparticles. When the solution was allowed to standfor over 2 hours, precipitation of large macroscopic aggregates wasobserved. A ‘melting analysis’ of the solution with the suspendedaggregates was performed. To perform the ‘melting analysis’, thesolution was diluted to 1 ml with hybridization buffer, and the opticalsignature of the aggregates at 260 nm was recorded at one minuteintervals as the temperature was increased from 25° C. to 75° C., with aholding time of 1 minute/degree. Consistent with characterization of theaggregate as an oligonucleotide-nanoparticle polymer, a characteristicsharp transition (full width at half maximum, FW_(1/2) of the firstderivative=3.5° C.) was observed with a “melting temperature” (T_(m)) of53.5° C. This compares well with the T_(m) associated with the broadertransition observed for oligonucleotides without nanoparticles(T_(m)=54° C., FW_(1/2)=˜13.5° C.). The ‘melting analysis’ of theoligonucleotide solution without nanoparticles was performed undersimilar conditions as the analysis with nanoparticles, except that thetemperature was increased from 10–80° C. Also, the solution was 1.04 μMin each oligonucleotide component.

To test the selectivity of the system, the T_(m) for the aggregateformed from the perfect complement 4 of probes 1 and 2 was compared withthe T_(m)'s for aggregates formed from targets that contained one basemismatches, deletions, or insertions (FIGS. 15A–G). Significantly, allof the gold nanoparticle-oligonucleotide aggregates that containedimperfect targets exhibited significant, measurable destabilization whencompared to the aggregates formed from the perfect complement, asevidenced by T_(m) values for the various aggregates (see FIGS. 15A–G).The solutions containing the imperfect targets could easily bedistinguished from the solution containing the perfect complement bytheir color when placed in a water bath held at 52.5° C. Thistemperature is above the T_(m) of the mismatched polynucleotides, soonly the solution with the perfect target exhibited a purple color atthis temperature. A ‘melting analysis’ was also performed on the probesolution which contained the half-complementary target. Only a minuteincrease in absorbance at 260 nm was observed.

Next, 2 μL (20 picomoles) of each of the oligonucleotide targets (FIGS.15A–G) were added to a solution containing 50 μL of each probe (13 nM)in hybridization buffer. After standing for 15 minutes at roomtemperature, the solutions were transferred to a temperature-controlledwater bath and incubated at the temperatures indicated in Table 4 belowfor five minutes. A 3 μl sample of each reaction mixture was thenspotted on a C-18 silica plate. Two control experiments were performedto demonstrate that the alignment of both probes onto the target isnecessary to trigger aggregation and, therefore, a color change. Thefirst control experiment consisted of both probes 1 and 2 without targetpresent. The second control experiment consisted of both probes 1 and 2with a target 3 that is complementary to only one of the probe sequences(FIG. 15B). The results are presented in Table 4 below. Pink spotssignify a negative test, and blue spots signify a positive test.

Notably, the colorimetric transition that can be detected by the nakedeye occurs over less than 1° C., thereby allowing one to easilydistinguish the perfect target 4 from the targets with mismatches (5 and6), an end deletion (7), and a one base insertion at the point in thetarget where the two oligonucleotide probes meet (8) (see Table 4). Notethat the colorimetric transition T_(c) is close in temperature, but notidentical, to T_(m). In both controls, there were no signs of particleaggregation or instability in the solutions, as evidenced by the pinkishred color which was observed at all temperatures, and they showednegative spots (pink) in the plate test at all temperatures (Table 4).

The observation that the one base insertion target 8 can bedifferentiated from the fully complementary target 4 is truly remarkablegiven the complete complementarity of the insertion strand with the twoprobe sequences. The destabilization of the aggregate formed from 8 andthe nanoparticle probes appears to be due to the use of two short probesand the loss of base stacking between the two thymidine bases where theprobe tails meet when hybridized to the fully complementary target. Asimilar effect was observed when a target containing a three base pairinsertion (CCC) was hybridized to the probes under comparableconditions, (T_(m)=51° C.). In the system described above in Example 5,targets with base insertions could not be distinguished from the fullycomplementary target. Therefore, the system described in this example isvery favorable in terms of selectivity. This system also exhibited thesame sensitivity as the system described in Example 5, which isapproximately 10 femtomoles without amplification techniques.

The results indicate that any one base mismatch along the target strandcan be detected, along with any insertions into the target strand.Importantly, the temperature range over which a color change can bedetected is extremely sharp, and the change occurs over a very narrowtemperature range. This sharp transition indicates that there is a largedegree of cooperativity in the melting process involving the largenetwork of colloids which are linked by the target oligonucleotidestrands. This leads to the remarkable selectivity as shown by the data.

TABLE 4 Reactants Results (probes) + (color) target RT 47.6° C. 50.5° C.51.4° C. 52.7° C. 54.5° C. (1 + 2) pink pink pink pink pink pink (1 +2) + 3 pink pink pink pink pink pink (1 + 2) + 4 blue blue blue blueblue pink (1 + 2) + 5 blue blue blue pink pink pink (1 + 2) + 6 bluepink pink pink pink pink (1 + 2) + 7 blue blue blue blue pink pink (1 +2) + 8 blue blue pink pink pink pink

Example 8 Assays Using Nanoparticle-Oligonucleotide Conjugates

A set of experiments were performed involving hybridization with‘filler’ duplex oligonucleotides. Nanoparticle-oligonucleotideconjugates 1 and 2 illustrated in FIG. 16A were incubated with targetsof different lengths (24, 48 and 72 bases in length) and complementaryfiller oligonucleotides, as illustrated in FIGS. 16A–C. Otherwise, theconditions were as described in Example 7. Also, the oligonucleotidesand nanoparticle-oligonucleotide conjugates were prepared as describedin Example 7.

As expected, the different reaction solutions had markedly differentoptical properties after hybridization due to the distance-dependentoptical properties of the gold nanoparticles. See Table 5 below.However, when these solutions were spotted onto a C-18 TLC plate, a bluecolor developed upon drying at room temperature or 80° C., regardless ofthe length of the target oligonucleotide and the distance between thegold nanoparticles. See Table 5. This probably occurs because the solidsupport enhances aggregation of the hybridizedoligonucleotide-nanoparticle conjugates. This demonstrates that byspotting solutions onto the TLC plate, the distance between the goldnanoparticles can be substantial (at least 72 bases), and colorimetricdetection is still possible.

TABLE 5 Results (Color) Target Length Solution TLC Plate 24 bases BlueBlue 48 bases Pink Blue 72 bases Pink Blue Probes 1 + 2 only Pink Pink

The color changes observed in this and other examples occur when thedistance between the gold nanoparticles (the interparticle distance) isapproximately the same or less than the diameter of the nanoparticle.Thus, the size of the nanoparticles, the size of the oligonucleotidesattached to them, and the spacing of the nanoparticles when they arehybridized to the target nucleic acid affect whether a color change willbe observable when the oligonucleotide-nanoparticle conjugates hybridizewith the nucleic acid targets to form aggregates. For instance, goldnanoparticles with diameters of 13 nm will produce a color change whenaggregated using oligonucleotides attached to the nanoparticles designedto hybridize with target sequences 10–35 nucleotides in length. Thespacing of the nanoparticles when they are hybridized to the targetnucleic acid adequate to give a color change will vary with the extentof aggregation, as the results demonstrate. The results also indicatethat the solid surface enhances further aggregation ofalready-aggregated samples, bringing the gold nanoparticles closertogether.

The color change observed with gold nanoparticles is attributable to ashift and broadening of the surface plasmon resonance of the gold. Thiscolor change is unlikely for gold nanoparticles less than about 4 nm indiameter because the lengths of the oligonucleotides necessary forspecific detection of nucleic acid would exceed the nanoparticlediameter.

Example 9 Assays Using Nanoparticle-Oligonucleotide Conjugates

Five microliters of each probe 1 and 2 (FIG. 12A) were combined to afinal concentration of 0.1 M NaCl with buffer (10 mM phosphate, pH 7),and 1 microliter of human urine was added to the solution. When thissolution was frozen, thawed, and then spotted on a C-18 TLC plate, ablue color did not develop. To a similar solution containing 12.5microliters of each probe and 2.5 microliters of human urine, 0.25microliters (10 picomoles) of target 3 (FIG. 12A) was added. Thesolution was frozen, thawed and then spotted onto a C-18 TLC plate, anda blue spot was obtained.

Similar experiments were performed in the presence of human saliva. Asolution containing 12.5 microliters of each probe 1 and 2 and 0.25microliters of target 3 was heated to 70° C. After cooling to roomtemperature, 2.5 microliters of a saliva solution (human saliva diluted1:10 with water) was added. After the resultant solution was frozen,thawed and then spotted onto a C-18 TLC plate, a blue spot was obtained,indicating hybridization of the probes with the target. In controlexperiments with no target added, blue spots were not observed.

Example 10 Assays Using Nanoparticle-Oligonucleotide Conjugates

An assay was performed as illustrated in FIG. 13A. First, glassmicroscope slides, purchased from Fisher scientific, were cut intoapproximately 5×15 mm pieces, using a diamond tipped scribing pen.Slides were cleaned by soaking for 20 minutes in a solution of 4:1H₂SO₄:H₂O₂ at 50° C. Slides were then rinsed with copious amounts ofwater, then ethanol, and dried under a stream of dry nitrogen.Thiol-modified DNA was adsorbed onto the slides using a modifiedprocedure reported in the literature (Chrisey et al., Nucleic AcidsRes., 24, 3031–3039 (1996) and Chrisey et al., Nucleic Acids Res., 24,3040–3047 (1996)). First, the slides were soaked in a 1% solution oftrimethoxysilylpropyldiethyltriamine (DETA, purchased from UnitedChemical Technologies, Bristol, Pa.) in 1 mM acetic acid in Nanopurewater for 20 minutes at room temperature. The slides were rinsed withwater, then ethanol. After drying with a dry nitrogen stream, the slideswere baked at 120° C. for 5 minutes using a temperature-controlledheating block. The slides were allowed to cool, then were soaked in a 1mM succinimidyl 4-(malemidophenyl)-butyrate (SMPB, purchased from SigmaChemicals) solution in 80:20 methanol:dimethoxysulfoxide for 2 hours atroom temperature. After removal from the SMPB solution and rinsing withethanol, amine sites that were not coupled to the SMPB crosslinker werecapped as follows. First, the slides were soaked for 5 minutes in a 8:1THF:pyridine solution containing 10% 1-methyl imidazole. Then the slideswere soaked in 9:1 THF:acetic anhydride solution for five minutes. Thesecapping solutions were purchased from Glen Research, Sterling, Va. Theslides were rinsed with THF, then ethanol, and finally water.

DNA was attached to the surfaces by soaking the modified glass slides ina 0.2 OD (1.7 μM) solution containing freshly purified oligonucleotide(3′ thiol ATGCTCAACTCT [SEQ ID NO:33]). After 12 hours of soaking time,the slides were removed and rinsed with water.

To demonstrate the ability of an analyte DNA strand to bindnanoparticles to the modified substrate, a linking oligonucleotide wasprepared. The linking oligonucleotide was 24 bp long (5′TACGAGTTGAGAATCCTGAATGCG [SEQ ID NO:34]) with a sequence containing a 12bp end that was complementary to the DNA already adsorbed onto thesubstrate surface. The substrate was then soaked in a hybridizationbuffer (0.5 M NaCl, 10 mM phosphate buffer pH 7) solution containing thelinking oligonucleotide (0.4 OD, 1.7 μM) for 12 hours. After removal andrinsing with similar buffer, the substrate was soaked in a solutioncontaining 13 nm diameter gold nanoparticles which had been modifiedwith an oligonucleotide (TAGGACTTACGC 5′ thiol [SEQ ID NO:35]) that iscomplementary to the unhybridized portion of the linking oligonucleotideattached to the substrate. After 12 hours of soaking, the substrate wasremoved and rinsed with the hybridization buffer. The glass substrate'scolor had changed from clear and colorless to a transparent pink color.See FIG. 19A.

Additional layers of nanoparticles were added to the slides by soakingthe slides in a solution of the linking oligonucleotide as describedabove and then soaking in a solution containing 13 nm gold nanoparticleshaving oligonucleotides (3′ thiol ATGCTCAACTCT [SEQ ID NO:33]) attachedthereto. After soaking for 12 hours, the slides were removed from thenanoparticle solution and rinsed and soaked in hybridization buffer asdescribed above. The color of the slide had become noticeably more red.See FIG. 19A. A final nanoparticle layer was added by repeating thelinking oligonucleotide and nanoparticle soaking procedures using 13 nmgold nanoparticles which had been modified with an oligonucleotide(TAGGACTTACGC 5′ thiol [SEQ ID NO:35]) as the final nanoparticle layer.Again, the color darkened, and the UV-vis absorbance at 520 nmincreased. See FIG. 19A.

To verify that the oligonucleotide modified gold nanoparticles wereattached to the oligonucleotide modified surface through DNAhybridization interactions with the linking oligonucleotide, a meltingcurve was performed. For the melting experiment, a slide was placed in acuvette containing 1.5 mL of hybridization buffer, and an apparatussimilar to that used in Example 2, part B, was used. The absorbancesignal due to the nanoparticles (520 nm) was monitored at each degree asthe temperature of the substrate was increased from 20° C. to 80° C.,with a hold time of 1 minute at each integral degree. The nanoparticlesignal dramatically dropped when the temperature passed 52° C. See FIG.19B. A first derivative of the signal showed a melting temperature of55° C., which corresponds with the temperature seen for theoligonucleotide-nanoparticle conjugates and linking oligonucleotideshybridized in solution. See FIG. 19B.

Example 11 Assay of a Polyribonucleotide UsingNanoparticle-Oligonucleotide Conjugates as Probes

The previous Examples utilized oligo-deoxyribonucleotides as targets inthe assays. The present example demonstrates that thenanoparticle-oligonucleotide conjugates can also be used as probes inassaying a polyribonucleotide. The experiment was carried out by adding1 μL of a solution of poly(rA) (0.004 A₂₆₀ Units) to 100 μL of goldnanoparticles (˜10 nM in particles) conjugated to dT₂₀ (a 20-meroligonucleotide containing thymidylate residues) through a mercaptoalkyllinker at the 5′-terminus. The conjugation procedure was that describedin Example 3. Following freezing in a Dry Ice/isopropyl alcohol bath,thawing at room temperature, and spotting on a C18 TLC plate asdescribed in Example 4, a blue spot characteristic of aggregation of thenanoparticles by hybridization was observed. Control experiments carriedout in absence of the target gave a pink spot, rather than a blue spot.

Example 12 Assay for Protective Antigen DNA Segment of Anthrax UsingNanoparticle-Oligonucleotide Conjugates

In many cases amplification of a double-stranded DNA target by PCR isneeded to provided sufficient material for an assay. The present exampledemonstrates that the nanoparticle-oligonucleotide conjugates can beused to assay for a DNA strand in the presence of its complement (i.e.,assaying for a single strand after thermal dehybridization of adouble-stranded target) and can recognize and specifically bind to anamplicon obtained from a PCR reaction.

A PCR solution containing a 141 base pair duplex amplicon of theProtective Antigen segment of Anthrax was provided by the Navy (sequencegiven in FIG. 23). The assay for this amplicon was carried out byisolating the DNA from 100 μL of the PCR solution using a QiaquickNucleotide Removal Kit (Qiagen, Inc., Santa Clarita, Calif.) and thestandard protocol for this kit, with the exception that elution of theDNA was effected with 10 mM phosphate buffer at pH 8.5, rather than withthe buffer provided with the kit. The eluant was then evaporated todryness on a Speed Vac (Savant). To this residue was added 5 μL of amaster mix prepared by mixing equal volumes of each of two solutions oftwo different oligonucleotide-nanoparticle probes (see FIG. 23). Eacholigonucleotide-nanoparticle probe was prepared as described in Example3. The solutions of the probes which were combined to form the mastermix were prepared by adding 10 μL of 2 M NaCl and 5 μL ofoligonucleotide blocker solution (50 pmoles of each Blockeroligonucleotide (see FIG. 23 and below) in a 0.3 M NaCl, 10 mMphosphate, pH 7.0., solution) to 5 μL of full-strength (about 10 nM)nanoparticle-oligonucleotide solution. The amplicon-probe mixture washeated to 100° C. for 3 minutes, then frozen in a DRY ICE/ethanol bathand allowed to come to room temperature. A small aliquot (2 μL) wasspotted on a C18 TLC plate and allowed to dry. A strong blue spotindicative of hybridization was obtained.

Control tests carried out in the same manner in absence of the amplicontarget DNA, in the absence of Probe 1, in the absence of Probe 2, or inthe absence of the sodium chloride, were all negative, that is, gave apink spot. Similarly a test carried out using probes 1 and 2 with a PCRamplicon derived from the Lethal Factor segment of Anthrax in place ofthe Protective Antigen Segment was negative (pink spot). These controlsconfirmed that both probes were essential, that salt conditionsappropriate for hybridization were needed, and that the test wasspecific for the specified target sequence.

The oligonucleotide Blockers were added to inhibit binding of the secondstrand of the initial duplex target (i.e., the strand complementary tothe target strand) to regions of the target nucleic acid strand outsidethe segment that binds to the probes (see FIG. 23 for sequences), sincesuch binding interferes with binding of the nanoparticle oligonucleotideprobes to the target strand. In this example, the Blockeroligonucleotides were complementary to the single-stranded target inregions not covered by the probes. An alternative scheme is to useblocker oligonucleotides that are complementary to the PCR complementarystrand (the strand complementary to the target strand) outside theregion that competes with the probe oligonucleotides.

Example 13 Direct Assay of PCR Amplicons without Isolation of theAmplicons from the PCR Solution

The procedure described in Example 12 involved separation of the PCRamplicon from the PCR solution before addition of thenanoparticle-oligonucleotide probes. For many purposes it would bedesirable to be able to carry out the assay directly in the PCR solutionwithout preliminary isolation of the polynucleotide products. A protocolfor such an assay has been developed and is described below. Thisprotocol has been performed successfully with several PCR productsderived under standard conditions using a GeneAmp PCR Reagent Kit withAmplitaq DNA polymerase.

To 50 μL of the PCR sample solution, 5 mL of a mixture of two goldnanoparticle-oligonucleotide probes (0.008 A₅₂₀ Units of each) wasadded, followed by addition of a solution made up from 1 μL of Blockeroligonucleotides (10 pmoles each), 5 μL of 5 M NaCl, and 2 μL of 150 mMMgCl₂. This mixture was heated for 2 minutes at 100° C. to separate thestrands of the duplex target, the tube was immersed directly in a coldbath (e.g., Dry Ice/ethanol) for 2 minutes, then removed, and thesolution allowed to thaw at room temperature (the freeze-thaw cyclefacilitates hybridization of the probes with the targetoligonucleotide). Finally, a few μL of the solution were spotted on aplate (e.g., C18 RP TLC plate, a silica plate, a nylon membrane, etc.).As usual, blue color signifies the presence of the targeted nucleic acidin the PCR solution; a pink color is negative for this target.

Example 14 Direct Recognition of Duplex Oligonucleotides WithoutDehybridization, Using Assembly of Nanoparticle-OligonucleotideConjugates

In the previous Examples, double-stranded targets were dehybridized byheating to generate single strands which interacted with single-strandedoligonucleotide probes bound to nanoparticles. The present exampledemonstrates that in cases where triple-stranded complexes can form,double-stranded oligonucleotide sequences can be recognized by thenanoparticle probes without prior dehybridization of the target.

Tests were carried out with two different systems—polyA:polyU anddA₄₀:dT₄₀—by adding 1 μL of a solution containing 0.8 A₂₆₀ Units of thetarget duplex in 100 μL of buffer (0.1 M NaCl, 10 mM phosphate, pH 7.0)to 100 μL of a colloidal solution of Au-sdT₂₀nanoparticle-oligonucleotide conjugate (˜10 nM in particles; see Example11) in 0.3 M NaCl, 10 mM phosphate buffer at pH 7.0. Subsequent quickfreezing by immersing the tube in a Dry Ice/isopropyl alcohol bath andthawing by removing the tube from the bath and letting it stand at roomtemperature (22° C.), followed by spotting 3 μL of the solution on a C18TLC plate, afforded a blue spot characteristic of hybridization andaggregation of the nanoparticles.

The rationale for this test is that the nanoparticle probes (bearingpyrimidine oligonucleotides in this example) bind in a sequence specificmanner at purine oligonucleotide/pyrimidine oligonucleotide sites alongthe duplex target. Since many binding sites are available on each doublestranded entity, the binding leads to formation of an aggregate ofnanoparticles. The results show that this assay, based on formation oftriple-stranded complexes involving the nanoparticle probes, works bothfor oligoribo- and oligodeoxyribonucleotide double-stranded targets.

Example 15 Assay Employing Both Fluorescence and Colorimetric Detection

All hybridization experiments were performed in a 0.3 M NaCl, 10 mMphosphate, pH 7.0, buffer solution. AcetatePlus™ filtration membranes(0.45 μm) were purchased from Micron Separations Inc., Westboro, Mass.Alkylamine-functionalized latex microspheres (3.1 μm) were purchasedfrom Bangs Laboratories, Fishers Ind. Fluorophore-labeledoligonucleotides functionalized with alklylamino groups at the3′-terminus were synthesized using standard phosphoramidite chemistry(Eckstein, ed., in Oligonucleotides and Analogues, 1 st ed., OxfordUniversity, New York, N.Y. 1991) with an Amino-Modifier C7 CPG solidsupport (Glen Research) and a 5′-fluorescein phosphoramidite (6-FAM,Glen Research) on an Expedite 8909 synthesizer and were purified byreverse phase HPLC. They were attached to the amine-functionalized latexmicrospheres by means of diisothiocyanate coupling to yield a dithiourealinkage as described in Charreyre et al., Langmuir, 13, 3103–3110(1997). Briefly, a DMF solution of a one thousand fold excess of1,4-phenylene diisothiocyanate was added to an aqueous borate buffersolution (0.1 M, pH 9.3) of the amino-modified oligonucleotide. Afterseveral hours, the excess 1,4-phenylene diisothiocyanate was extractedwith butanol and the aqueous solution lyophilized. The activatedoligonucleotides were redissolved in borate buffer and reacted with theamino-functionalized latex microspheres in a carbonate buffer (0.1 M, pH9.3, 1 M NaCl). After 12 hrs, the particles were isolated bycentrifugation and washed three times with buffered saline solution (0.3M NaCl, 10 mM phosphate pH 7.0). The 5′-oligonucleotide-modified goldnanoparticle probes were prepared as described in Example 3.

The target oligonucleotide (1–5 μl, 3 nM) was added to 3 μl offluorophore-labeled oligonucleotide-modified latex microsphere probesolution (3.1 μm; 100 fM). After 5 minutes, 3 μl of the 5′oligonucleotide-modified gold nanoparticle probe solution (13 nm; 8 nM)were added to the solution containing the target and latex microsphereprobes. Upon standing for an additional 10 minutes, the solutioncontaining both probes and target was vacuum-filtered through theAcetatePlus membrane. The membrane retained the relatively large latexparticles and allowed any non-hybridized gold nanoparticle probes topass through. In the presence of a sufficient concentration of target,the latex microspheres and the gold nanoparticles hybridized with thetarget, and a red spot was observed on the membrane (positive result). Acontrol experiment was always carried out where the aliquot of solutioncontaining the target oligonucleotide was replaced by an equal volume ofwater. In this case, a white spot was left on the membrane (negativeresult). For a 24-base-pair model system, using the unaided eye, 3femtomoles of target oligonucleotide could be detected colorimetrically.

A double-stranded target oligonucleotide (1–5 μl, 20 nM), 3 μl of asolution of fluorophore-labeled-oligonucleotide-latex microspheres (3.1μm; 100 fM) and 3 μl of a solution of 5′-oligonucleotide-goldnanoparticles (13 nm; 8 nM) were combined and heated to 100° C. for 3minutes. Then, the solution was immediately frozen by immersing thereaction vessel containing it in a liquid N₂ bath for 3 minutes. Thissolution was then thawed at room temperature and filtered as describedabove. For a 24-base pair model system, using the unaided eye, 20femtomoles of duplex target oligonucleotide could be detectedcolorimetrically.

When monitored by fluorescence, the detection method described aboveproved to be difficult due to background fluorescence from the membrane.This problem was overcome by “washing” the latex microspheres bycentrifugation to remove excess gold nanoparticle probes before spottingan aliquot on a reverse-phase TLC plate. The hybridization experimentswere performed as described above. After hybridization was effectedbetween the probes and target, 10 μl of buffer were added to thesolution, which was subsequently centrifuged at 10,000×g for 2 minutes.The supernatant was removed, and 5 μl of buffer were added to helpresuspend the precipitate. A 3 μL aliquot was then spotted on areverse-phase TLC plate. For both single-stranded and duplex targetoligonucleotides, 25 femtomoles could be detected colorimetrically bythe naked eye. Fluorescent spots could be visualized by the naked eyewith a hand-held UV-lamp until the target amount in the 3 μl aliquotused to form the spot was as low as 50 femtomoles. It is believed thatoptimization of this system will allow for detection of even loweramounts of target nucleic acid.

Example 16 Assays Employing Silver Staining

DNA hybridization tests on oligonucleotide-modified substrates arecommonly used to detect the presence of specific DNA sequences insolution. The developing promise of combinatorial DNA arrays for probinggenetic information illustrates the importance of these heterogeneoussequence assays to future science. In most assays, the hybridization offluorophore-labeled targets to surface-bound probes is monitored byfluorescence microscopy or densitometry. Although fluorescence detectionis very sensitive, its use is limited by the expense of the experimentalequipment and by background emissions from most common substrates. Inaddition, the selectivity of labeled oligonucleotide targets forperfectly complementary probes over those with single-base mismatches ispoor, preventing the use of surface hybridization tests for detection ofsingle nucleotide polymorphisms. A detection scheme which improved uponthe simplicity, sensitivity and selectivity of fluorescent methods couldallow the full potential of combinatorial sequence analysis to berealized. The present invention provides such improved detectionschemes.

For instance, oligonucleotide-modified gold nanoparticles and unmodifiedDNA target could be hybridized to oligonucleotide probes attached to aglass substrate in a three-component sandwich assay (see FIGS. 25A–B).Note that the nanoparticles can either be individual ones (see FIG. 25A)or “trees” of nanoparticles (see FIG. 25B). The “trees” increase signalsensitivity as compared to the individual nanoparticles, and thehybridized gold nanoparticles “trees” often can be observed with thenaked eye as dark areas on the glass substrate. When “trees” are notused, or to amplify the signal produced by the “trees,” the hybridizedgold nanoparticles can be treated with a silver staining solution. The“trees” accelerate the staining process, making detection of targetnucleic acid faster as compared to individual nanoparticles.

The following is a description of one specific system (illustrated inFIG. 25A). Capture oligonucleotides (3′-HS(CH₂)₃-A₁₀ATGCTCAACTCT; SEQ IDNO: 43) were immobilized on a glass substrate as described in Example10. A target oligonucleotide (5′-TACGAGTTGAGAATCCTGAATGCG-3′, SEQ ID NO:44, concentrations given below in Table 6 for each experiment) washybridized with the capture oligonucleotides in 0.3 M NaCl, 10 mMphosphate buffer as described in Example 10. The substrate was rinsedtwice with the same buffer solution and immersed in a solutioncontaining gold nanoparticle probes functionalized withtarget-complementary DNA (5′-HS(CH₂)₆A₁₀CGCATTCAGGAT, SEQ ID NO:45)(preparation described in Example 3) for 12 hours. Next, thesubstrate was rinsed copiously with 0.3 M NaNO₃ to remove Cl⁻. Thesubstrate was then developed with silver staining solution (1:1 mixtureof Silver Enhancer Solutions A and B, Sigma Chemical Co., # S-5020 and #S-5145) for 3 minutes. Greyscale measurements were made by scanning thesubstrate on a flatbed scanner (normally used for scanning documentsinto a computer) linked to a computer loaded with software capable ofcalculating greyscale measurements (e.g., Adobe Photoshop). The resultsare presented in Table 6 below.

TABLE 6 Target DNA Con- centration Mean Greyscale Standard Deviation 10nM 47.27 2.10 5 nM 53.45 0.94 2 nM 54.56 1.17 1 nM 59.98 1.82 500 pM61.61 2.26 200 pM 90.06 3.71 100 pM 99.04 2.84 50 pM 135.20 7.49 20 pM155.39 3.66 None 168.16 10.03 (control)

Example 17 Assemblies Containing Quantum Dots

This example describes the immobilization of synthetic single-strandedDNA on semiconductor nanoparticle quantum dots (QDs). Native CdSe/ZnScore/shell QDs (˜4 nm) are soluble only in organic media, making directreaction with alkylthiol-terminated single-stranded DNA difficult. Thisproblem was circumvented by first capping the QDs with3-mercaptopropionic acid. The carboxylic acid group was thendeprotonated with 4-(dimethylamino)pyridine, rendering the particleswater soluble, and facilitating reaction of the QDs with either3′-propylthiol- or 5′-hexylthiol-modified oligonucleotide sequences.After DNA modification, the particles were separated from unreacted DNAby dialysis. A “linker” DNA strand was then hybridized to surface-boundsequences, generating extended assemblies of nanoparticles. The QDassemblies, which were characterized by TEM, UV/Visible spectroscopy,and fluorescence spectroscopy, could be reversibly assembled bycontrolling the temperature of the solution. The temperature dependentUV-Vis spectra were obtained for the novel QD assemblies and compositeaggregates formed between QDs and gold nanoparticles (˜13 nm).

A. General Methods

Nanopure water (18.1 MΩ) prepared using a NANOpure ultrapure waterpurification system was employed throughout. Fluorescence spectra wereobtained using a Perkin Elmer LS 50 B Luminescence Spectrometer. Meltinganalyses were performed using a HP 8453 diode array spectrophotometerequipped with a HP 9090a Peltier Temperature Controller. Centrifugationwas carried out using either an Eppendorf 5415C centrifuge or a BeckmanAvanti 30 centrifuge. TEM images were acquired using a Hitachi HF-2000field emission TEM operating at 200 kV.

B. Preparation of Oligonucleotide-QD Conjugates

Synthetic methodologies for semiconductor quantum dots (QDs) haveimproved greatly in recent years, and for some materials, most notablyCdSe, monodisperse samples of pre-determined size can now be preparedwith relative ease. Murray et al., J. Am. Chem. Soc. 1993, 115, 8706;Hines, et al., J. Phys. Chem. 1996, 100, 468. As a result, the uniqueelectronic and luminescent properties of these particles have beenstudied extensively (see, Alivisatos, J. Phys. Chem. 1996, 100, 13226,and references therein; Klein et al., Nature 1997, 699; Kuno et al., J.Chem. Phys. 1997, 106, 9869; Nirmal et al., Nature 1996, 383, 802),potentially paving the way for QDs to be employed in diversetechnologies, such as light-emitting diodes (Schlamp et al., J. Appl.Phys. 1997, 82, 5837; Dabbousi et al., Appl. Phys. Lett. 1995, 66, 1316)and as non-radioactive biological labels (Bruchez et al., Science 1998,281, 2013; Chan et al., Science 1998, 281, 2016). However, manyapplications will require that the particles be arranged spatially on asurface or organized into three-dimensional materials (Vossmeyer et al.,J. Appl. Phys. 1998, 84, 3664). Moreover, the ability to organize one ormore types of nanoparticles into superlattice structures (Murray et al.,Science 1995, 270, 1335) would allow for the construction of completelynew types of hybrid materials with new and potentially interesting anduseful properties.

DNA is the ideal synthon for programming the assembly of nanoscalebuilding blocks into periodic two- and three-dimensional extendedstructures. The many attributes of DNA, which include ease of synthesis,extraordinary binding specificity, and virtually unlimitedprogrammability by virtue of nucleotide sequence, can be exploited forthe use of QD assembly.

The modification of QDs with DNA has proven to be more difficult thanfor gold nanoparticles. The common methods for preparing highlyluminescent CdSe QDs yield materials that are coated with a mixture oftrioctylphosphine oxide (TOPO) and trioctylphosphine (TOP). As a result,these QDs are soluble only in non-polar solvents, making them difficultto functionalize with highly charged DNA strands by direct reaction.This difficulty has been overcome by the method described below, whichis the first successful modification of semiconductor nanoparticles withsingle-stranded DNA. It should be noted that others, in elegant studies,have looked at the interactions between QDs and duplex DNA, but thesestudies did not make use of the sequence specific binding properties ofDNA to direct the assembly of extended QD structures. Coffer et al.,Appl. Phys. Lett, 1996, 69, 3851; Mahtab et al., J. Am. Chem. Soc.,1996, 118, 7028.

Since the surface of CdSe/ZnS core/shell QDs binds organic thiols, itwas desired to modify these semiconductor particles withalkylthiol-terminated DNA strands by a substitution reaction. The lackof water solubility of these QDs, though, hindered such an approach. Twodifferent methods recently have been reported for making QDs watersoluble, allowing for the immobilization of protein structures on the QDsurfaces. One involves encapsulation of the core/shell structures with asilica layer (Bruchez et al., Science 1998, 281, 2013), while the otherutilizes mercaptoacetic acid both to stabilize the particles and providewater solubility (Chan et al., Science 1998, 281, 2016). The proceduredescribed in this example, which produces remarkably stable colloidunder DNA hybridization conditions, utilizes 3-mercaptopropionic acid topassivate the QD surface.

An excess of 3-mercaptopropionic acid (0.10 mL, 1.15 mmol; Aldrich) wasadded by syringe to a suspension of −20 mg of TOP/TOPO stabilizedCdSe/ZnS QDs (prepared as described in Hines, et al., J. Phys. Chem.1996, 100, 468) in 1.0 mL of N,N,-dimethyl formamide (DMF; Aldrich)generating a clear, dark orange solution containing 3-mercaptopropionicacid functionalized QDs. The reaction occurred quickly. For subsequentreactions, excess 3-mercaptopropionic acid was not removed, and theparticles were stored at room temperature in DMF.

However, to characterize the QDs, a portion of the sample was purifiedby removing unreacted 3-mercapto-propionic acid as follows. A 0.50 mLsample was centrifuged (4 hours at 30,000 rpm), and the supernatant wasremoved. The remaining solution was washed with ˜0.3 mL of DMF andrecentrifuged. This step was repeated two additional times beforerecording the FTIR spectrum. FTIR (polyethylene card, 3M): 1710 cm⁻¹(s), 1472 cm⁻¹ (m), 1278 cm⁻¹ (w), 1189 cm⁻¹ (m), 1045 cm⁻¹ (w), 993cm⁻¹ (m), 946 cm⁻¹ (w), 776 cm⁻¹ (m), 671 cm⁻¹ (m). Unlike the TOP/TOPOstabilized native QDs, the 3-mercaptopropionic acid modified QDsexhibited a characteristic v_(CO) band at 1710 cm⁻¹ for the surfacebound propionic acid.

Although the 3-mercaptopropionic acid modified QDs were practicallyinsoluble in water, their solubility could be significantly enhanced bydeprotonating the surface bound mercaptopropionic acid sites with4-(dimethylamino)pyridine (DMAP; Aldrich) as described in the nextparagraph. The QDs then dispersed readily in water, producing orangesolutions that were stable for up to a week at room temperature.

To attach oligonucleotides to QDs, 150 μL (optical density at 530nm=21.4) of a solution of the 3-mercaptopropionic acid functionalizedparticles in DMF were added to a solution of DMAP (8.0 mg, 0.065 nmol)in 0.4 mL of DMF. An orange precipitate was formed. It was separated bycentrifugation (−30 seconds at 3000 rpm) and then dissolved in 1.0 mL ofa solution of 3′ propylthiol- or 5′ hexylthiol-terminatedoligonucleotides (1.0–2.0 ODs/mL; prepared as described in Example 1;sequences given below). Precipitate (dissolved in water) wascharacterized by IR spectroscopy (polyethylene card, 3M). IR (cm⁻¹):1647 (m), 1559 (s), 1462 (m), 1214 (w), 719 (w), 478 (s). After standingfor 12 hours, the oligonucleotide-containing solution was brought to0.15 M NaCl, and the particles were aged for an additional 12 hours. TheNaCl concentration was then raised to 0.3 M, and the mixture was allowedto stand for a further 24–40 hours before dialyzing against PBS (0.3 MNaCl, 10 mM phosphate buffer, pH 7, 0.01% sodium azide) using a 100 kDamembrane (Spectra/Por Cellulose Ester Membrane). The dialysis wascarried out over a period of 48 hours, during which time the dialysisbath was refreshed three times.

Oligonucleotide-QD conjugates prepared in this manner displayedindefinite aqueous stability. Moreover, the colloid remained stronglyfluorescent, with a sharp [full width at half maximum (FWHM)=33 nm],symmetrical emission at 546 mm (indicative of a ˜3.2 nm CdSe core;Murray et al., J. Am. Chem. Soc. 1993, 115, 8706).

Two different oligonucleotide-QD conjugates were prepared by thisprotocol and stored in PBS. One was modified with a 22mer, comprised ofa propylthiol functionality at the 3′-end, a 12mer capture sequence, andan intervening 10 base (all A) spacer: 5′-TCTCAACTCGTAA₁₀-(CH₂)₃-SH [SEQID NO: 46]. The other employed a 5′-hexylthiol-terminated sequence, alsowith a 10 base (all A) spacer, and a 12mer capture sequence which wasnon-complementary with the 3′-propylthiol sequence:5′-SH-(CH₂)₆-A₁₀CGCATTCAGGAT-3′ [SEQ ID NO: 47].

C. Preparation of OD Assemblies

When approximately equal quantities of these two oligonucleotides (200μL, OD₅₃₀=0.224 and 0.206, respectively) were mixed and then combinedwith 6 μL (60 pmol) of a solution of a complementary linking 24mersequence (5′-TACGAGTTGAGAATCCTGAATGCG-3′, SEQ ID NO: 48), QD assembliesformed within 20–30 minutes at room temperature, FIG. 26. Faster linkingtook place when the mixture was frozen (−78° C.) and then allowed towarm slowly to room temperature.

The clusters generated were not large enough to settle out of solution.However, they could be separated by centrifugation at relatively lowspeeds (10,000 RPM for 10 min), as compared with the unlinked particles(30,000 RPM for 2–3 hours).

The decrease in fluorescence upon hybridization was determined byintegration of the fluorescence signal (320 nm excitation wavelength)from 475 nm to 625 nm of 4 pairs of samples. Each pair was prepared inthe following manner. A solution of of 3′ propylthiol-terminatedDNA-modified particles (30 μL, optical density at 530 nm=0.224) wascombined with a solution of 5′ hexylthiol-terminated DNA-modified QDs(30 μL, optical density at 530 nm=0.206) in an Eppendorf centrifugetube, and then diluted with 140 μL of PBS. The mixture was then splitinto two equal portions, and complementary “linker” DNA (3 μL, 30 pmol)was added to one, while non-complementary “linker” DNA(5′-CTACTTAGATCCGAGTGCCCACAT-3′, SEQ ID NO:49) (3 μL, 30 pmol) was addedto the other. All eight of the samples were then frozen in a dryice/acetone bath (−78° C.), after which they were removed from the bathand allowed to warm slowly to room temperature. To estimate the changein fluorescence efficiency upon hybridization, the fluorescenceintensities of the “target” (complementary “linker”) samples wereadjusted to account for the difference in absorbance at 320 nm from thecorresponding control samples, which contained non-complementary“linker”.

The results showed that hybridization of QD/QD assemblies wasaccompanied by a decrease in integrated fluorescence intensity by anaverage of 26.4±6.1%, and a ˜2 nm red shift of the emission maximum,presumably due to cooperative effects between QDs, FIG. 27A.Interestingly, Bawendi, et al. noticed a similar, albeit slightlylarger, red shift when comparing the fluorescence of close-packed QDsand widely separated dots isolated in a frozen matrix (Murray et al.,Science 1995, 270, 1335). These changes in the fluorescence spectra maybe an indication of excimer formation between QDs, but the exact natureof such a complex is still largely a matter of speculation. As expected,no aggregation was observed when the “linker” was missing or notcomplementary, or when either one of the two types of particles wasabsent.

The “melting” behavior of the DNA was monitored by observing the UV-Visspectra of the aggregates as a function of temperature. For this“melting” analysis, the precipitate containing the QD/QD assemblies wascentrifuged at 10,000 rpm for 10 minutes, washed with 7 μL of PBS,recentrifuged, and suspended in 0.7 mL of PBS. The UV/Visiblespectroscopic signature of the assemblies was recorded at two degreeintervals as the temperature was increased from 25° C. to 75° C., with aholding time of 1 minute prior to each measurement. The mixture wasstirred at a rate of 500 rpm to ensure homogeneity throughout theexperiment. Temperature vs extinction profiles were then compiled fromthe extinction at 600 nm. The first derivative of these profiles wasused to determine the “melting” temperatures.

The results, FIG. 27B (T_(m)57° C.), demonstrated conclusively that DNAhad been immobilized on the QD surfaces and that hybridization wasresponsible for the assembly process. The transition also was extremelysharp when compared with DNA alone (FWHM of the respective firstderivatives: 4° C. vs 9° C.), which is consistent with the formation ofan aggregate structure with multiple DNA links per particle. An increasein extinction was observed upon denaturation, most likely because of ascreening effect whereby particles in the interiors of the assembliesare prevented from absorbing light by the surrounding QDs.

D. Preparation of QD/Gold Assemblies

With DNA-functionalized QDs in hand, the assembly of hybrid assembliesmade from multiple types of nanoparticle building blocks becamefeasible. To prepare these hybrid assemblies, a solution of ˜17 nM3′-hexylthiol-modified 13 nm gold nanoparticles (30 μL, ˜5 fmol;prepared as described in Example 3) was mixed with a solution of5′-hexylthiol-terminated DNA-modified QDs (15 μL, optical density at 530nm=0.206) in an Eppendorf centrifuge tube. “Linker” DNA (5 μL, 50 pmol)was added, and the mixture cooled to −78° C., and then allowed to warmslowly to room temperature, generating a reddish-purple precipitate. Noaggregation behavior was observed unless both types of particles and acomplementary target were present. After centrifugation (1 min at 3,000rpm) and removal of the supernatant, the precipitate was washed with 100μL of PBS and recentrifuged.

For “melting” analysis, the washed precipitate was suspended in 0.7 mLof PBS. UV-Vis spectroscopy was used to follow the changes in thesurface plasmon resonance of the gold nanoparticles, so temperature vs.extinction profiles were compiled at 525 nm. Using the surface plasmonresonance of the gold nanoparticles provides a much more sensitive probewith which to monitor hybridization than does the UV-Vis spectroscopicsignature of the QDs alone. Therefore, a “melting” experiment can beperformed on a much smaller sample (˜10% of the QD solution is needed),although the intensity of the plasmon band obscures the UV/Vis signalfrom the QDs. Similar to the pure QD system described above, a sharp(FWHM of the first derivative=4.5° C.) melting transition occurred at58° C. (see FIG. 27D).

High resolution TEM images of these assemblies showed a network of goldnanoparticles interconnected by multiple QDs, FIG. 27C. The QDs, whichhave a much lower contrast in the TEM image than gold nanoparticles, canbe identified by their lattice fringes. They are just barely resolvablewith the high resolution TEM, but clearly indicate the periodicstructure of these composite assemblies and the role that DNA plays informing them.

E. Summary

The results described in this example definitively establish that theimmobilization of DNA onto QD surfaces has been achieved and that theseparticles can now be used in combination with DNA under hybridizationconditions. Using DNA-functionalized QDs, the first DNA-directedformation of QD and mixed gold/QD nanoparticle structures has beendemonstrated. The successful modification of semiconductor QDs with DNAhas significant implications for materials research, and the door is nowopen for more extensive inquiries into the luminescent, electronic, andchemical properties of these unique building blocks as they areincorporated into new and functional multi-component nanostructures andnanoscale materials.

Example 18 Methods of Synthesizing Oligonucleotide-NanoparticleConjugates and the Conjugates Produced by the Methods A. General Methods

HAuCl₄-3H₂O and trisodium citrate were purchased from Aldrich chemicalcompany, Milwaukee, Wis. Gold wire, 99.999% pure, and titanium wire werepurchased from Goldsmith Inc., Evanston, Ill. Silicon wafers (100) witha 1 micron thick oxide layer were purchased from Silicon QuestInternational, Santa Clara, Calif. 5′-thiol-modifier C6-phosphoramiditereagent, 3′-propylthiol modifier CPG, fluorescein phosphoramidite, andother reagents required for oligonucleotide synthesis were purchasedfrom Glen Research, Sterling, Va. All oligonucleotides were preparedusing an automated DNA synthesizer (Expedite) using standardphosphoramidite chemistry (Eckstein, F. Oligonucleotides and Analogues;1st ed.; Oxford University Press, New York, 1991). Oligonucleotidescontaining only 5′ hexylthiol modifications were prepared as describedin Example 1. 5-(and 6)-carboxyfluorescein, succinimidyl ester waspurchased from Molecular Probes, Eugene, Oreg. NAP-5 columns (SephadexG-25 Medium, DNA grade) were purchased from Pharmacia Biotech. NanopureH₂O (>18.0 MΩ), purified using a Barnstead NANO pure ultrapure watersystem, was used for all experiments. An Eppendorf 5415C or a BeckmanAvanti 30 centrifuge was used for centrifugation of Au nanoparticlesolutions. High Performance Liquid Chromatography (HPLC) was performedusing a HP series 1100 HPLC.

B. Physical Measurements

Electronic absorption spectra of the oligonucleotide and nanoparticlesolutions were recorded using a Hewlett-Packard (HP) 8452a diode arrayspectrophotometer. Fluorescence spectroscopy was performed using aPerkin-Elmer LS50 fluorimeter. Transmission Electron Microscopy (TEM)was performed with a Hitachi 8100 Transmission Electron Microscopeoperating at 200 kV. A Thermo Jarrell Ash AtomScan 25 atomic emissionspectrometer with an inductively coupled plasma (ICP) source was used todetermine the atomic concentration of gold in the nanoparticle solutions(gold emission was monitored at 242.795 nm).

C. Synthesis and Purification of Fluorescein-LabeledAlkanethiol-Modified Oligonucleotides

Thiol-modified oligonucleotide strands containing either 12 or 32 bases,with 5′ hexylthiol and 3′ fluorescein moieties, were prepared. Thesequence of the 12mer (S12F) was HS(CH₂)₆-5′-CGC-ATT-CAG-GAT-3′-(CH₂)₆-F[SEQ ID NO:50], and the 32mer (SA₂₀12F) contained the same 12mersequence with the addition of a 20 dA spacer sequence to the 5′ end [SEQID NO:51]. The thiol-modified oligonucleotides were prepared asdescribed in Storhoff et al., J. Am. Chem. Soc. 120:1959–1964 (1998). Anamino-modifier C7 CPG solid support was used in automated synthesis, andthe 5′ terminus was manually modified with hexylthiol phosphoramidite,as described previously. The 3′ amino, 5′ trityl-protected thiolmodified oligonucleotides were purified by reverse-phase HPLC using anHP ODS Hypersil column (5 mm, 250×4 mm) with 0.03 M triethyl ammoniumacetate (TEAA), pH 7 and a 1%/minute gradient of 95% CH₃CN/5% 0.03 MTEAA at a flow rate of 1 mL/min., while monitoring the UV signal of DNAat 254 nm. The retention times of the 5′-S-trityl, 3′ amino modified12-base and 32-base oligonucleotides were 36 and 32 minutesrespectively.

The lyophilized product was redispersed in 1 ml of 0.1 M Na₂CO₃ and,while stirring in the dark, 100 μL of 10 mg/ml succinimidyl ester offluorescein (5,6 FAM-SE, Molecular Probes) in dry DMF was added over 1.5hours according to the directions of the manufacturer (Molecular Probesliterature). The solution was stirred at room temperature for anadditional 15 hours, then precipitated from 100% ethanol at −20° C. Theprecipitate was collected by centrifugation, dissolved in H₂O and thecoupled product separated from unreacted amino-terminatedoligonucleotide by ion-exchange HPLC. A Dionex Nucleopac PA-100 column(250×4 mm) was operated with 10 mM NaOH aqueous eluent and a 1%/minutegradient of 1 M NaCl/10 mM NaOH at a flow rate of 0.8 mL/minute.Retention times of 5′-S-trityl, 3′ fluorescein modified 12mer and 32merwere 50 and 49 minutes respectively. The oligonucleotide product wasdesalted by reverse-phase HPLC. Removal of the trityl protection groupof the fluorescein-terminated, trityloligonucleotide was performed usingsilver nitrate and dithiothreitol (DTT) as previously described(Storhoff et al., J. Am. Chem. Soc. 120:1959–1964 (1998)). The yield andpurity of the oligonucleotides were assessed using the techniquespreviously described for alkylthiol oligonucleotides (Storhoff et al.,J. Am. Chem. Soc. 120:1959–1964 (1998)). Oligonucleotides were usedimmediately after detritylation of the thiol group.

Thiol-modified oligonucleotides containing 32 bases, with 3′ propylthioland 5′ fluorescein moieties(HS)CH₂)₃-3′-(W)₂₀-TAG-GAC-TTA-CGC-5′-(CH₂)₆-F, W=A or T) [SEQ ID NO:52]were synthesized on an automated synthesizer using 3′ thiol modifierCPG. The 5′ terminus of each oligonucleotide was coupled manually tofluorescein phosphoramidite (6-FAM, Glen Research). The modifiedoligonucleotides were purified by ion exchange HPLC (1%/min gradient of1 M NaCl, 10 mM NaOH; retention time (Rt)˜48 min (W=T), Rt˜29 min(W=A)). After purification, the oligonucleotide solutions were desaltedby reverse phase HPLC. The 3′ thiol moieties were deprotected withdithiothreitol by a procedure previously described (Storhoff et al., J.Am. Chem. Soc. 120:1959–1964 (1998)).

D. Synthesis and Purification of Fluorescein Labeled Oligonucleotides

The fluorophore labeled complement (12° F.) consisted of 12 bases3′-GCG-TAA-GTC-CTA-5′-(CH₂)₆—F [SEQ ID NO:53] complementary to the 12mersequence in S12F and SA₂₀12F. The oligonucleotide was synthesized usingstandard methods, and a syringe-based procedure, similar to theprocedure reported above for the 5′ alkylthiol modification, was used tocouple fluorescein phosphoramidite (6-FAM, Glen Research) to the 5′ endof the CPG-bound oligonucleotide. Purification was performed usingreverse-phase HPLC as above. The fluorescein-labeled oligonucleotide hada retention time of 18 min. The fluorophore labeled complement, 3′12F(5′-ATC-CTG-AAT-GCG-F; [SEQ ID NO:54]) was prepared using anamino-modifier C7 CPG solid support for automated synthesis, followed bycoupling of 5-(6)-carboxyfluorescein succinimidyl ester to the 3′ amineusing the procedure described above.

E. Preparation and Characterization of Gold Nanoparticles

Gold nanoparticles were prepared by citrate reduction of HAuCl₄ asdescribed in Example 1. Transmission Electron Microscopy (TEM) performedwith a Hitachi 8100 TEM was used to determine the size distribution ofthe resulting nanoparticles. At least 250 particles were sized from TEMnegatives using graphics software (ImageTool). The average diameter of atypical particle preparation was 15.7±1.2 nm. Assuming sphericalnanoparticles and density equivalent to that of bulk gold (19.30 g/cm²),an average molecular weight per particle was calculated (2.4×10⁷ g/mol).The atomic gold concentration in a solution of gold nanoparticles wasdetermined by ICP-AES (inductively coupled plasmon atomic emissionspectroscopy). A gold atomic absorption standard solution (Aldrich) wasused for calibration. Comparison of atomic gold concentration in theparticle solution to the average particle volume obtained by TEManalysis yielded the molar concentration of gold particles in a givenpreparation, typically ˜10 nM. By measuring the UV-vis absorbance ofnanoparticle solutions at the surface plasmon frequency (520 nm), themolar extinction coefficients (ε at 520 nm) were calculated for theparticles, typically 4.2×10⁸ M⁻¹ cm⁻¹ for 15.7±1.2 nm diameterparticles.

F. Preparation of Gold Thin Films

Silicon wafers were cut into ˜10 mm×6 mm pieces and cleaned with piranhaetch solution (4:1 concentrated H₂SO₄: 30% H₂O₂) for 30 min at 50° C.,then rinsed with copious amounts of water, followed by ethanol.(Warning: piranha etch solution reacts violently with organic materialsand should be handled with extreme caution.) Metal was deposited at arate of 0.2 nm/s using an Edwards Auto 306 evaporator (base pressure of3×10⁻⁷ millibar) equipped with an Edwards FTM6 quartz crystalmicrobalance. The oxidized sides of the silicon were coated with a Tiadhesion layer of 5 nm, followed by 200 nm of gold.

G. Preparation of 5′ Alkylthiol Oligonucleotide-Modified GoldNanoparticles

Gold nanoparticles were modified with fluorescein-alkylthiololigonucleotides by adding freshly deprotected oligonucleotides toaqueous nanoparticle solution (particle concentration ˜10 nM) to a finaloligonucleotide concentration of 3 μM. After 24 hours, the solution wasbuffered at pH 7 (0.01 M phosphate), and NaCl solution was added (tofinal concentration of 0.1 M). The solution was allowed to ‘age’ underthese conditions for an additional 40 hours. Excess reagents were thenremoved by centrifugation for 30 minutes at 14,000 rpm. Followingremoval of the supernatant, the red oily precipitate was washed twicewith 0.3 M NaCl, 10 mM phosphate buffer, pH 7, solution (PBS) bysuccessive centrifugation and redispersion, then finally redispersed infresh buffer solution. Invariably, a small amount (˜10% as determined byUV-vis spectroscopy) of nanoparticle is discarded with the supernatantduring the washing procedure. Therefore, final nanoparticleconcentrations were determined by TEM, ICP-AES, and UV-vis spectroscopy(see above). Extinction coefficients and particle size distributions didnot change significantly as a result of the oligonucleotidemodification.

H. Preparation of 5′ Alkylthiol Oligonucleotide-Modified Gold Thin Films

Silicon supported gold thin films were immersed in deposition solutionsof deprotected alkylthiol modified oligonucleotides for equal times andbuffer conditions as for the gold nanoparticles. Followingoligonucleotide deposition, the films were rinsed extensively with 0.3 MPBS and stored in buffer solution. Gold was evaporated on one side only,leaving an unpassivated silicon/silicon oxide face. However, alkylthiolmodified DNA did not adsorb appreciably to bare silicon oxide surfacesthat were rinsed with PBS.

I. Quantitation of Alkylthiol-Oligonucleotides Loaded on Nanoparticles

Mercaptoethanol (ME) was added (final concentration 12 mM) tofluorophore-labeled oligonucleotide modified nanoparticles or thin filmsin 0.3 M PBS, to displace the oligonucleotides. After 18 hours at roomtemperature with intermittent shaking, the solutions containingdisplaced oligonucleotides were separated from the gold by eithercentrifugation of the gold nanoparticles, or by removal of the gold thinfilm. Aliquots of the supernatant were diluted two-fold by addition of0.3 M PBS, pH 7. Care was taken to keep the pH and ionic strength of thesample and calibration standard solutions the same for all measurementsdue to the sensitivity of the optical properties of fluorescein to theseconditions (Zhao et al., Spectrochimica Acta 45A: 1113–1116 (1989)). Thefluorescence maxima (measured at 520 nm) were converted to molarconcentrations of the fluorescein-alkylthiol modified oligonucleotide byinterpolation from a standard linear calibration curve. Standard curveswere prepared with known concentrations of fluorophore-labeledoligonucleotides using identical buffer and salt concentrations.Finally, the average number of oligonucleotides per particle wasobtained by dividing the measured oligonucleotide molar concentration bythe original Au nanoparticle concentration. Normalized surface coveragevalues were then calculated by dividing by the estimated particlesurface area (assuming spherical particles) in the nanoparticlesolution. The assumption of roundness is based on a calculated averageroundness factor of 0.93. Roundness factor is computed as:(4×pi×Area)/perimeter×2) taken from Baxes, Gregory, Digital ImageProcessing, p. 157 (1994).

J. Quantitation of the Hybridized Target Surface Density

To determine the activity of attached oligonucleotides forhybridization, fluorophore-labeled oligonucleotides, which werecomplementary to the surface-bound oligonucleotides (12′F), were reactedwith oligonucleotide modified surfaces (gold nanoparticles or thinfilms) under hybridization conditions (3 μM complementaryoligonucleotide, 0.3 M PBS, pH 7, 24 hr). Non-hybridizedoligonucleotides were removed from the gold by rinsing twice withbuffered saline as described above. Then, the fluorophore-labeledoligonucleotides were dehybridized by addition of NaOH (finalconcentration ˜50 mM, pH 11–12, 4 hr). Following separation of thesolution containing the 12′F from the nanoparticle solutions bycentrifugation, and neutralization of the solutions by addition of 1 MHCl, the concentrations of hybridized oligonucleotide and correspondinghybridized target surface density were determined by fluorescencespectroscopy.

K. Quantitation of Surface Coverage and Hybridization

Citrate stabilized gold nanoparticles were functionalized with 12merfluorescein-modified alkylthiol DNA(HS-(CH₂)₆-5′-CGC-ATT-CAG-GAT-(CH₂)₄-F [SEQ ID NO:50]). Surface coveragestudies were then performed by thoroughly rinsing away non-chemisorbedoligonucleotides, followed by removal of the fluorophore-labeledoligonucleotides from the gold surface, and quantitation ofoligonucleotide concentration using fluorescence spectroscopy (asdescribed above).

Removal of all the oligonucleotides from the gold surface and subsequentremoval of gold nanoparticles from the solution is critical forobtaining accurate coverage data by fluorescence for several reasons.First, the fluorescence signal of labeled, surface bound DNA isefficiently quenched as a result of fluorescence resonance energytransfer (FRET) to the gold nanoparticle. Indeed, there is almost nomeasurable signal for fluorescein-modified oligonucleotides (12–32nucleotide strands, sequences are given above) after they areimmobilized on 15.7±1.2 nm gold nanoparticles and residualoligonucleotide in solution is washed away. Second, the goldnanoparticles absorb a significant amount of light between 200 nm and530 nm, so their presence in solution during fluorescence measurementsacts as a filter and diminishes the available excitation energy, as wellas the intensity of emitted radiation. The gold surface plasmon band at520 nm falls at the emission maximum of fluorescein.

Mercaptoethanol (ME) was used to rapidly displace the surface boundoligonucleotides by an exchange reaction. To examine the displacementkinetics, oligonucleotide-modified nanoparticles were exposed to ME (12mM) for increasing periods of time prior to centrifugation andfluorescence measurements. The intensity of fluorescence associated withthe solution free of nanoparticles can be used to determine how mucholigonucleotide was released from the nanoparticles. The amount ofoligonucleotide freed in exchange with ME increased until about 10 hoursof exposure (FIG. 29), which is indicative of complete oligonucleotidedisplacement. The displacement reaction was rapid, which is presumablydue to the inability of the oligonucleotide film to block access of theME to the gold surface (Biebuyck et al., Langmuir 9:1766 (1993)).

The average oligonucleotide surface coverage of alkylthiol-modified12mer oligonucleotide (S12F) on gold nanoparticles was 34±1 pmol/cm²(average of ten independent measurements of the sample.) For 15.7±1.2 nmdiameter particles, this corresponds to roughly 159 thiol-bound 12merstrands per gold particle. Despite slight particle diameter variationfrom batch to batch, the area-normalized surface coverages were similarfor different nanoparticle preparations.

In order to verify that this method is useful for obtaining accurateoligonucleotide surface coverages, it was used to displaceflourophore-labeled oligonucleotides from gold thin films, and thesurface coverage data was compared with experiments aimed at gettingsimilar information but with different techniques. In these experiments,gold thin films were subjected to a similar oligonucleotide modificationand ME displacement procedure as the citrate stabilized goldnanoparticles (see above). The oligonucleotide displacement versus timecurves for the gold thin films are very similar to those measured forgold nanoparticles. This suggests a similar rate of displacement for thethin films, even though the typical surface coverage values measured forthese films were somewhat lower than the oligonucleotide coverages ongold nanoparticles. Importantly, the oligonucleotide surface coverageson gold thin films measured by our technique (18±3 pmol/cm²) fall withinthe range of previously reported coverages on oligonucleotide thin films(10 pmol/cm² for a 25 base oligonucleotide on gold electrodes determinedusing electrochemistry or surface plasmon resonance spectroscopy (SPRS)(Steel et al., Anal. Chem. 70:4670–4677 (1998)). Differences in surfacecoverages are expected due to different oligonucleotide sequences andlengths, as well as film preparation methods.

The extent of hybridization of complementary fluorophore-labeledoligonucleotides (12F′) to nanoparticles with surface-bound 12meroligonucleotides was measured as described above. Briefly, S12F modifiednanoparticles were exposed to 12F′ at a concentration of 3 μM for 24hours under hybridization conditions (0.3 M PBS, pH 7) and then rinsedextensively with buffer solution. Again, it was necessary to remove thehybridized strands from the gold before measuring fluorescence. This wasaccomplished by denaturing the duplex DNA in a high pH solution (NaOH,pH 11) followed by centrifugation. Hybridized 12′F amounted to 1.3±0.2pmol/cm² (approximately 6 duplexes per 15.7 nm particle; the averagenumber of duplexes per particle was computed by multiplying thenormalized hybridized surface coverage in pmol/cm² by the averageparticle surface area as found from size distributions measured byTEM.). In order to measure the extent of non-specific adsorption, S12Fmodified gold nanoparticles were exposed to fluorophore-labelednon-complementary 12 base oligonucleotides (12F′) in 0.3 M PBS. Afterextensive rinsing (successive centrifugation/redispersion steps) andsubsequent high pH treatment, the coverage of non-specifically adsorbedoligonucleotides on the nanoparticles was determined to be on the orderof 0.1 pmol/cm². An analogous procedure was used to measurehybridization to S12F modified gold thin films in order to compare thehybridization results to reported values on gold electrodes. The degreeof hybridization, 6±2 pmol/cm², was consistent with hybridizationreported for mixed base 25mer on an gold electrode (2–6 pmol/cm²) (Steelet al., Anal. Chem. 70:4670–4677 (1998)).

Surface coverages and hybridization values of the S12F/12F′ system forboth nanoparticles and thin films are summarized in Table 7. The moststriking result is the low hybridization efficiency (˜4% ofsurface-bound strands on nanoparticles while 33% of strands on thinfilms hybridize). Previous studies have shown similarly lowhybridization for sufficiently densely packed oligonucleotidemonolayers. This may reflect a low accessibility to incoming hybridizingstrands, due to a combination of steric crowding of the bases,especially those near the gold surface, as well as electrostaticrepulsive interactions.

L. Effect of Oligonucleotide Spacer on Surface Coverage andHybridization

Although the high coverage of the S12F oligonucleotide is advantageousin terms of nanoparticle stabilization, the low hybridization efficiencyprompted us to devise a means of decreasing steric congestion around thehybridizing sequence. Oligonucleotides (32mer) were synthesized having a20 dA spacer sequence inserted between the alkylthiol group and theoriginal 12 base recognition sequence. This strategy was chosen based onthe assumption that: 1) bases near the nanoparticle surface aresterically inaccessible because of weak interactions between thenitrogenous bases and the gold surface, as well as interstrand stericcrowding, and 2) on a 15.7 nm diameter roughly spherical particle, 12mersequences attached to the end of 20mer spacer units roughlyperpendicular to the surface (Levicky et al., J. Am. Chem. Soc.120:9787–9792 (1998)) will lead to a film with a greater free volume ascompared with a film formed from the same 12mer directly bound to thesurface.

While the surface density of single-stranded SA₂₀12F strands (15±4pmol/cm²) was lower than that of S12F (34±1 pmol/cm²), the particlesmodified with a 32-mer using the identical surface modification showedcomparable stability compared to those modified with 12-mer. Asanticipated, the hybridization efficiency of the SA₂₀12F/12F′ system(6.6±0.2 pmol/cm², 44%) was increased to approximately 10 times that ofthe original S12F/12F′ system, Table 7.

M. Effect of Electrolyte Concentration During Oligonucleotide Attachment

In working with the S12F sequence a salt aging step was found to becrucial in obtaining stable oligonucleotide modified nanoparticles (seeExample 3). The gold nanoparticles modified with S12F in pure waterfused together irreversibly to form a black precipitate uponcentrifugation, while those aged in salt resisted aggregation whencentrifuged, even in high ionic strength solutions. It is proposed thatthe increased stability is due to higher oligonucleotide surfacecoverages which leads to greater steric and electrostatic protection.Using the SA₂₀12F modified particles, the effect of electrolyteconditions on oligonucleotide surface loading was investigated. As shownin Table 8, final surface coverages for gold nanoparticles which wereexposed to oligonucleotides in water for 48 hours are much lower(7.9±0.2 pmol/cm²) compared to those that were ‘aged’ in salt, orprepared by increasing the salt concentration gradually over the courseof the final 24 hours of the experiment (see above).

It is important to note that gold nanoparticles as synthesizedirreversibly agglomerate even in very low ionic strength media. Indeed,they are naturally incompatible with salts and especially polyanionssuch as oligonucleotides. This aging treatment is essential forpreparing stable oligonucleotide particles. Therefore, the particlesmust be initially modified with alkylthiol oligonucleotides in waterprior to gradually increasing the ionic strength. It is likely thatoligonucleotides initially lie flat, bound through weak interactions ofthe nitrogenous bases with gold. A similar mode of interaction has beenproposed for oligonucleotides on thin films (Herne et al., J. Am. Chem.Soc. 119:8916–8920 (1997)). However, the interaction betweenoligonucleotides and the positively charged nanoparticle surface (Weitzet al., Surf Sci. 158:147–164 (1985)) is expected to be even stronger.In the aging step, the high ionic strength medium effectively screenscharge repulsion between neighboring oligonucleotides, as well as,attraction between the polyanionic oligonucleotide and the positivelycharged gold surface. This allows more oligonucleotides to bind to thenanoparticle surface, thereby increasing oligonucleotide surfacecoverage.

N. Effect of Oligonucleotide Spacer Sequence on Surface Coverage

In order to examine how the sequence of the spacer affectsoligonucleotide coverage on Au nanoparticles, fluorescein-modified32-mer strands, with 20 dA and 20 dT spacers inserted between a 3′propylthiol and the fluorescein-labeled 12-mer sequence, were prepared.The most notable result of surface coverage and hybridization studies ofnanoparticles modified with S3′T₂₀12F and S3′A₂₀12F is the greatersurface coverage achieved with the 20 dT spacer (35±1 pmol/cm²), incomparison to the 20 dA spacer (24±1 pmol/cm²). The number of hybridizedstrands was comparable, although the percentage of surface bound strandswhich hybridized was lower for ST₂₀12mer nanoparticles (79%) than theSA₂₀12 nanoparticles (˜94%). These results suggest that dT richoligonucleotide strands interact non-specifically with the nanoparticlesurface to a lesser degree than dA rich oligonucleotide strands.Consequently, 20dT spacer segments may extend perpendicular from thegold surface, promoting higher surface coverages, while 20dA spacersegments block gold sites by lying flat on the particle surface.

O. Effect of Coadsorbed Diluent Oligonucleotides

In addition to efficient hybridization, another important property ofoligonucleotide modified nanoparticles is the possibility of adjustingthe total number of hybridization events. This is most readilyaccomplished by adjusting the surface density of recognition strands.Other researchers have used coadsorbed diluent alkylthiols such asmercaptohexanol with modified oligonucleotides on gold electrodes tocontrol hybridization (Steel et al., Anal. Chem. 70:4670–4677 (1998);Herne et al., J. Am. Chem. Soc. 119:8916–8920 (1997)). However, theinherent low stability of unprotected gold nanoparticles poses seriousconstraints on the choice of diluent molecule. A thiol modified 20 dAsequence (SA₂₀) [SEQ ID NO:55] proved to be suitable in terms ofmaintaining particle stability in the high ionic strength buffers whichare needed for hybridization and protecting the surface from nonspecificadsorption.

Nanoparticles were modified using solutions containing differentrecognition strand (SA₂₀12F) to diluent (SA₂₀) strand molar ratios. Theresulting particles were analyzed by the fluorescence method describedabove to determine the SA₂₀12F surface density, and then tested forhybridization efficiency with 12′F.

The SA₂₀12F surface density increased linearly with respect to theproportion of SA₂₀12F to SA₂₀ in the deposition solution, FIG. 30. Thisis an interesting result because it suggests that the ratio of SA₂₀12Fto SA₂₀ attached to the nanoparticles reflects that of the solution.This result is in contrast to what is normally seen for mixtures ofshort chain alkyl or ω-functionalized thiols, where solubility and chainlength play a crucial role in adsorption kinetics (Bain et al., J. Am.Chem. Soc. 111:7155–7164 (1989); Bain et al., J. Am. Chem. Soc.111:7164–7175 (1989)).

The amount of complementary 12F′ oligonucleotide which hybridized toeach different sample also increased linearly with increasing SA₂₀12Fsurface coverage, FIG. 31. The fact that this relationship is welldefined indicates that it is possible to predict and control the extentof hybridization of the nanoparticle-oligonucleotide conjugates. Thissuggests that hybridization of 12F′ becomes more difficult at higherSA₂₀12F coverages, which is most likely a result of steric crowding andelectrostatic repulsion between oligonucleotides.

P. Summary

This study has shown that it is important to achieve a balance betweenoligonucleotide coverage high enough to stabilize the nanoparticles towhich they are attached, yet low enough so that a high percentage of thestrands are accessible for hybridization with oligonucleotides insolution. This has been achieved by adjusting salt conditions duringoligonucleotide attachment to the nanoparticles to gain higholigonucleotide surface coverages, oligonucleotide spacer segments toreduce electrosteric interactions, and coadsorbed diluent strands toreproducibly control the average number of hybridization events for eachnanoparticle. It has also been shown that the nature of the tether(spacer) sequence influences the number of oligonucleotide strandsloaded onto gold nanoparticles. This work has important implicationsregarding understanding interactions between oligonucleotides andnanoparticles, as well as optimizing the sensitivity ofnanoparticle-oligonucleotide detection methods.

TABLE 7 Single strand surface coverage and corresponding hybridizedsurface coverages for gold thin films and gold nanoparticles. Comparisonbetween S12F and SA₂₀12F surface coverage and hybridization. Thiolmodified oligonucleotides were attached to the gold from 3 μM aqueoussolutions and aged in 0.1 M NaCl. All hybridization studies wereperformed in 0.3 M PBS, pH 7. Surface Coverage Hybridization Coverage %Hybridization Oligonucleotide Pair (pmol/cm²) (pmol/cm²) Efficiency Aunanoparticles S12F/12F′ 34 ± 1 1.3 ± 0.2 ~4% SA₂₀12F/12F′ 15 ± 4 6.6 ±0.2 ~44% Au thin films S12F/12F′ 18 ± 3 6 ± 2 ~33%

TABLE 8

Effect of salt aging on surface coverage of SA₂₀12F oligonucleotides togold nanoparticles and hybridization to 12F′. All hybridizationexperiments were performed in 0.3 M PBS, pH 7. Buffer conditions duringSurface Coverage Hybridization Coverage Hybridization adsorption ofalkylthiol DNA (pmol/cm²) (pmol/cm²) Efficiency (%) H₂O  7.9 ± 0.2 —^(a)— 0.1 M NaCl, 10 mM phosphate 15 ± 4 6.6 ± 0.2 ~44 1.0 M NaCl, 10 mMphosphate 20 ± 2 6.5 ± 0.2 ~33 ^(a)Reliable values for these experimentscould not be obtained due to a small amount of particle aggregationwhich occurred after centrifugation.

TABLE 9 Effect of oligonucleotide spacer sequence on surface coverageand hybridization efficiency. Hybridization Surface Coverage CoverageHybridization Oligonucleotide Pair (pmol/cm²) (pmol/cm²) Efficiency (%)S3′A₂₀12F/3′12F 24 ± 1  9 ± 2 ~38 S3′T₂₀12F/3′12F 35 ± 1 12 ± 1 ~34S3′A₂₀12F/S3′T₂₀12F = HS(CH₂)₃-3′-W₂₀-TAG-GAC-TTA-CGC-5′-(CH₂)₆-F [SEQID NO:52] 3′12F = 5′-ATC-CTG-AAT-GCG-F [SEQ ID NO:54]

Example 19 Gene Chip Assay

An ultraselective and ultrasensitive method for analyzing combinatorialDNA arrays using oligonucleotide-functionalized gold nanoparticles isdescribed in this example. An unusually narrow temperature range forthermal dissociation of nanoparticle-target complexes permits thediscrimination of a given oligonucleotide sequence from targets withsingle nucleotide mismatches with extraordinary selectivity. Inaddition, when coupled with signal amplification method based onnanoparticle-catalyzed reduction of silver (I), the sensitivity of thisnanoparticle array detection system exceeds that of the analogous,conventional fluorophore system by two orders of magnitude.

Sequence-selective DNA detection has become increasingly important asscientists unravel the genetic basis of disease and use this newinformation to improve medical diagnosis and treatment. Commonly usedheterogeneous DNA sequence detection systems, such as Southern blots andcombinatorial DNA chips, rely on the specific hybridization ofsurface-bound, single-strand capture oligonucleotides complementary totarget DNAs. Both the specificity and sensitivity of these assays aredependent upon the dissociation properties of capture strands hybridizedto perfectly-matched and mismatched targets. As described below, it hassurprisingly been discovered that a single type of nanoparticleshybridized to a substrate exhibits a melting profile that issubstantially sharper than both the analogous fluorophore-based systemand unlabeled DNA. Moreover, the melting temperature for thenanoparticle duplex is 11 degrees higher than for the analogousfluorophore system with identical sequences. These two observations,combined with the development of a quantitative signal amplificationmethod based upon nanoparticle catalyzed reduction of silver (I), haveallowed the development of a new chip-based detection system for DNAthat has single-base mismatch selectivity and a sensitivity that is twoorders of magnitude more sensitive than the conventional analogousfluorescence-based assays.

Gold nanoparticles (13 nm diameter) having oligonucleotide attached tothem prepared as described in Example 3 were used to indicate thepresence of a particular DNA sequence hybridized to a transparentsubstrate in a three-component sandwich assay format (see FIG. 32). In atypical experiment, a substrate was fabricated by functionalizing afloat glass microscope slide (Fisher Scientific) with amine-modifiedprobe oligonucleotides as described in Example 10. This method was usedto generate slides functionalized with a single type of oligonucleotidesover their entire surface or in arrays of multiple types ofoligonucleotides spotted with a commercial microarrayer. Nanoparticleshaving indicator oligonucleotides attached to them and synthetic 30-meroligonucleotide targets (based on the anthrax protective antigensequence) were then cohybridized to these substrates (see FIG. 32).Therefore, the presence of nanoparticles at the surface indicated thedetection of a particular 30-base sequence. At high targetconcentrations (≧1 nM), the high density of hybridized nanoparticles onthe surface made the surface appear light pink (see FIG. 33). At lowertarget concentrations, attached nanoparticles could not be visualizedwith the naked eye (although they could be imaged by field-emissionscanning electron microscopy). In order to facilitate the visualizationof nanoparticles hybridized to the substrate surface, a signalamplification method in which silver ions are catalytically reduced byhydroquinone to form silver metal on the slide surface was employed.Although this method has been used for enlargement of protein- andantibody-conjugated gold nanoparticles in histochemical microscopystudies (Hacker, in Colloidal Gold: Principles, Methods, andApplications, M. A. Hayat, Ed. (Academic Press, San Diego, 1989), vol.1, chap. 10; Zehbe et al., Am. J. Pathol. 150, 1553 (1997)) its use inquantitative DNA hybridization assays is novel (Tomlinson et al., Anal.Biochem., 171:217 (1988)). Not only did this method allow very lowsurface coverages of nanoparticle probes to be visualized by a simpleflatbed scanner or the naked eye (FIG. 33), it also permittedquantification of target hybridization based on the optical density ofthe stained area (FIG. 34). Significantly, in the absence of the target,or in the presence of noncomplementary target, no staining of thesurface was observed, demonstrating that neither nonspecific binding ofnanoparticles to the surface, nor nonspecific silver staining, occurs.This result is an extraordinary feature of thesenanoparticle-oligonucleotide conjugates which enables ultra-sensitiveand -selective detection of nucleic aicds.

It has been determined that the unique hybridization properties ofoligonucleotide-functionalized nanoparticles of the present inventioncan be further used to improve the selectivity of combinatorialoligonucleotide arrays (or “gene chips”) (Fodor, Science 277, 393(1997)). The relative ratio of target hybridized to different elementsof an oligonucleotide array will determine the accuracy of the array indetermining the target sequence; this ratio is dependent upon thehybridization properties of the duplex formed between different capturestrands and the DNA target. Remarkably, these hybridization propertiesare dramatically improved by the use of nanoparticle labels instead offluorophore labels. As shown in FIG. 35, the dehybridization ofnanoparticle-labeled targets from surface-bound capture strands was muchmore sensitive to temperature than that of fluorophore-labeled targetswith identical sequences. While the fluorophore-labeled targetsdehybridized from surface capture strands over a very broad temperaturerange (first derivative FWHM=16° C.), identical nanoparticle-labeledtargets melted much more sharply (first derivative FWHM=3° C.). It wasanticipated that these sharpened dissociation profiles would improve thestringency of chip-based sequence analysis, which is usually effected bya post-hybridization stringency wash. Indeed, the ratio of targethybridized to complementary surface probes to that hybridized tomismatched probes after a stringency wash at a specific temperature(represented by the vertical lines in FIG. 35) is much higher withnanoparticle labels than fluorophore labels. This should translate tohigher selectivity in chip detection formats. In addition, nanoparticlelabels should increase array sensitivity by raising the meltingtemperature (T_(m)) of surface duplexes, which lowers the criticalconcentration below which duplexes spontaneously melt at roomtemperature.

In order to evaluate the effectiveness of nanoparticles as calorimetricindicators for oligonucleotide arrays, test chips were probed with asynthetic target and labeled with both fluorophore and nanoparticleindicators. The test arrays and oligonucleotide target were fabricatedaccording to published protocols (Guo et al., Nucl. Acids Res., 22:5456(1994); arrays of 175 μm diameter spots separated by 375 μm werepatterned using a Genetic Microsystems 417 Microarrayer). Arrayscontained four elements corresponding to the each of the four possiblenucleotides (N) at position 8 of the target (see FIG. 32). The synthetictarget and either fluorescent-labeled or nanoparticle-labeled probeswere hybridized stepwise to arrays in hybridization buffer, and eachstep was followed with a stringency buffer wash at 35° C. First, 20 μLof a 1 nM solution of synthetic target in 2×PBS (0.3 M NaCl, 10 mMNaH₂PO₄/Na₂HPO₄ buffer, pH 7) was hybridized to the array for 4 hours atroom temperature in a hybridization chamber (Grace Bio-Labs Cover WellPC20), and then washed at 35° C. with clean 2×PBS buffer. Next, 20 μL ofa 100 pM solution of oligonucleotide-functionalized gold nanoparticlesin 2×PBS was hybridized to the array for 4 hours at room temperature ina fresh hybridization chamber. The array was washed at 35° C. with clean2×PBS, then twice with 2×PBN (0.3 M NaNO₃, 10 mM NaH₂PO₄/Na₂HPO₄ buffer,pH 7). Then, the nanoparticle arrays were immersed in a silveramplification solution (Sigma Chemical, Silver Enhancer Solution) for 5min and washed with water. Silver amplification darkened the arrayelements considerably, and 200 μm diameter elements could be easilyimaged with a flatbed scanner or even the naked eye.

Arrays challenged with the model target and nanoparticle-labeled probesand stained with the silver solution clearly exhibited highly selectivehybridization to complementary array elements (FIG. 36A). Redundantspots of the same capture sequence showed reproducible and consistenthybridization signal. No background adsorption by nanoparticles orsilver stain was observed; the image greyscale value reported by theflatbed scanner is the same as that observed for a clear microscopeslide. The darker spots corresponding to adenine at position 8 (N=A)indicate that oligonucleotide target hybridized preferentially toperfectly complementary capture strands over mismatched ones, by agreater than 3:1 ratio. In addition, integrated greyscale values foreach set of spots follows the predicted stability of the Watson-Crickbase pairs, A:T>G:T>C:T>T:T (Allawi et al., Biochemistry 36, 10581,(1988)). Normally, G:T mismatches are particularly difficult todiscriminate from A:T complements (Saiki et al., in Mutation Defection,Cotton et al., eds. (Oxford University Press, Oxford, 1998), chap. 7; S.Ikuta et al., Nucl. Acids Res. 15, 797 (1987)), and the distinction ofthese two array elements demonstrates the remarkable resolving power ofnanoparticle labels in single nucleotide mismatch detection. Theselectivity of the nanoparticle-based arrays was higher than that of thefluorophore-indicated arrays, FIG. 36B; fluorophore labels provided only2:1 selectivity for adenine at position 8.

The assays utilizing nanoparticle-labeled probes were significantly moresensitive than those utilizing fluorophore-labeled probes. Hybridizationsignal could be resolved at the N=A elements at target concentrations aslow as 50 fM (or, for a hybridization chamber containing 20 μL ofsolution, 1×10⁶ total copies); this represents a dramatic increase insensitivity over common Cy3/Cy5 fluorophore-labeled arrays, for which ˜1pM or greater target concentrations are typically required. The highermelting temperatures observed for nanoparticle-target complexesimmobilized on surfaces undoubtedly contribute to array sensitivity. Thegreater stability of the probe/target/surface-oligonucleotide complex inthe case of the nanoparticle system as compared with the fluorophoresystem presumably results in less target and probe lost during washingsteps.

Colorimetric, nanoparticle labeling of combinatorial oligonucleotidearrays will be useful in applications such as single nucleotidepolymorphism analysis, where single mismatch resolution, sensitivity,cost and ease of use are important factors. Moreover, the sensitivity ofthis system, which has yet to be totally optimized, points toward apotential method for detecting oligonucleotide targets without the needfor target amplification schemes such as polymerase chain reaction.

Example 20 Nanoparticle Structures

The reversible assembly of supramolecular layered gold nanoparticlestructures onto glass supports, mediated by hybridized DNA linkers, isdescribed. Layers of oligonucleotide-functionalized nanoparticles weresuccessively attached to oligonucleotide-functionalized glass substratesin the presence of a complementary DNA linker. The unique recognitionproperties of DNA allow the nanoparticle structures to be assembledselectively in the presence of the complementary linker. In addition,the structures can be assembled and disassembled in response to externalstimuli which mediate hybridization of the linking duplex DNA, includingsolution temperature, pH, and ionic strength. In addition to offering avery selective and controlled way of building nanoparticle basedarchitectures on a solid support, this system allows one to study thefactors that influence both the optical and melting properties ofnanoparticle network structures linked with DNA.

Others have demonstrated how bifunctional organic molecules (Gittins etal., Adv. Mater. 11:737 (1999); Brust et al., Langmuir 14:5425 (1998);Bright et al., Langmuir 14:5695 (1998); Grabar et al., J. Am. Chem. Soc.118:1148 (1996); Freeman et al., Science 267:1629 (1995); Schmid et al.,Angew. Chem. Int. Ed. Engl. 39:181 (2000); Marinakos et al., Chem.Mater. 10:1214 (1998)) or polyelectrolytes (Storhoff et al., J. Am.Chem. Soc. 120:1959 (1998); Storhoff et al., J. Cluster Sci. 8:179(1997); Elghanian et al., Science 277:1078 (1997); Mirkin et al., Nature382:607 (1996)) can be used to controllably construct mono- andmultilayered nanoparticle materials off of planar substrates. Theattractive feature of using DNA as a nanoparticle interconnect is thatone can synthetically program interparticle distances, particleperiodicities, and particle compositions through choice of DNA sequence.Moreover, one can utilize the reversible binding properties ofoligonucleotides to ensure the formation of thermodyanamic rather thankinetic structures. In addition to providing a new and powerful methodfor controlling the growth of nanoparticle-based architectures fromsolid substrates, this strategy also allows one to evaluate therelationship between nanoparticle aggregate size and both melting andoptical properties of aggregate DNA-interlinked structures. Anunderstanding of these two physical parameters and their relationship tomaterials architecture is essential for utilizing nanoparticle networkmaterials, especially in the area of biodetection.

The oligonucleotide-functionalized, 13-nm-diameter gold nanoparticlesused to construct the multilayer assemblies were prepared as describedin Examples 1 and 3. The nanoparticles had 5′-hexanethiol-cappedoligonucleotide 1 (5′-HS(CH₂)₆O(PO₂ ⁻)O-CGCATTCAGGAT-3′ [SEQ ID NO:50])and 3′-propanethiol-capped oligonucleotide 2 (3′-HS(CH₂)₃)O(PO₂⁻)O-ATGCTCAACTCT-5′ [SEQ ID NO:59]) attached to them to yieldnanoparticles a and b, respectively (see FIG. 37). Glass slides werefunctionalized with 12-mer oligonucleotide 2 as described in Example 10.To build nanoparticle layers, the substrates were first immersed in a 10nM solution of 24-mer linker 3 (5′-TACGAGTTGAGAATCCTGAATGCG-3′ [SEQ INNO:60]) and allowed to hybridize with it for 4 hours at room temperature(see FIG. 37). The substrates were washed with clean buffer solution,and then hybridized with a 2 nM solution of particle a for 4 hours atroom temperature to attach the first nanoparticle layer. A secondnanoparticle layer could be attached to the first one by similarlyexposing the surface to solutions of linker 3 and nanoparticle b. Thesehybridization steps could be repeated to attach multiple, alternatinglayers of nanoparticles a and b, each layer connected to the previousone by linker 3. In the absence of linker, or in the presence ofnoncomplementary oligonucleotide, no hybridization of nanoparticles tothe surface was observed. In addition, multilayer assembly was onlyobserved under conditions which promoted the hybridization of the DNAlinkers: neutral pH, moderate salt concentration (>0.05 M NaCl), and atemperature below the duplex melting temperature (T_(m)).

Each hybridized nanoparticle layer imparted a deeper red color to thesubstrate, and after ten hybridized layers, the supporting glass slideappeared reflective and gold in color. Transmission UV-vis spectroscopyof the substrate was used to monitor the successive hybridization ofnanoparticle layers to the surface, FIG. 38A. The low absorbance of theinitial nanoparticle layer suggests that it seeded the formation offurther layers, which showed a near linear increase in the intensity ofthe plasmon band with each additional layer (for each successivenanoparticle layer formation, no additional absorbance was observed onexposure for longer times or to higher concentrations of either linker 3or nanoparticle solution). The linearity of the absorbance increaseafter the generation of the initial nanoparticle layer indicates thatthe surface was saturated with hybridized nanoparticles with eachsuccessive application, FIG. 38B. This is supported by field-emissionscanning electron microscope (FE-SEM) images of one (FIG. 39A) and two(FIG. 39B) nanoparticle layers on a surface, which show low nanoparticlecoverage with one layer, but near complete coverage with two layers. Theλ_(max) of the plasmon band for the multilayer assemblies shifts no morethan 10 nm, even after 5 layers. The direction of this shift isconsistent with other experimental (Grabar et al., J. Am. Chem. Soc.118:1148 (1996)) and theoretical (Quinten et al., Surf. Sci. 172:557(1986); Yang et al., J. Chem. Phys. 103:869 (1995)) treatments of goldnanoparticle aggregates. However, the magnitude of the shift is smallcompared to that previously observed for suspensions ofoligonucleotide-linked gold nanoparticle networks, which show maxλ_(max)>570 nm (see previous examples). This suggests that many morelinked nanoparticles—perhaps hundreds or thousands—are required toproduce the dramatic color change from red to blue observed for goldnanoparticle-based oligonucleotide probes. (Storhoff et al., J. Am.Chem. Soc. 120:1959 (1998); Storhoff et al., J. Cluster Sci. 8:179(1997); Elghanian et al., Science 277:1078 (1997); Mirkin et al., Nature382:607 (1996).). Surface plasmon shifts for aggregated goldnanoparticles have been shown to be highly dependent on interparticledistance (Quinten et al., Surf. Sci. 172:557 (1986); Storhoff et al., J.Am. Chem. Soc., in press), and the large distances provided byoligonucleotide linkers (8.2 nm for this system)) significantly reducethe progressive effect of nanoparticle aggregation on the gold surfaceplasmon band.

The dissociation properties of the assembled nanoparticle multilayerswere highly dependent upon the number of layers. When themultilayer-coated substrates were suspended in buffer solution and thetemperature raised above the T_(m) of the linking oligonucleotides (53°C.), the nanoparticles dissociated into solution, leaving behind acolorless glass surface. Increasing or decreasing the pH (>11 or <3) ordecreasing the salt concentration of the buffer suspension (below ˜0.01M NaCl) also dissociated the nanoparticles by dehybridizing the linkingDNA. The multilayer assembly was fully reversible, and nanoparticlescould be hybridized to, and dehybridized from, the glass substrates(e.g. three cycles were demonstrated with no detectable irreversiblenanoparticle binding).

Significantly, while all of the surface bound nanoparticle assembliesdissociated above the T_(m) of the linking oligonucleotides, thesharpness of these transitions depended on the size of the supportedaggregate, FIGS. 39D–F. Surprisingly, the dissociation of the firstnanoparticle layer from the substrate exhibited a transition (FIG. 39D,FWHM of the first derivative=5° C.) that was sharper than that of thesame oligonucleotides without nanoparticles in solution, FIG. 39C. Asmore nanoparticle layers were hybridized to the substrate, the meltingtransition of the oligonucleotide-linked nanoparticles becamesuccessively sharper (FIGS. 39E–F, FWHM of the first derivative=3° C.),until it matched that of the large nanoparticle network assemblies foundin solution. (Gittins et al., Adv. Mater. 11:737 (1999); Brust et al.Langmuir 14:5425 (1998)). These experiments confirm that more than twonanoparticles and multiple DNA interconnects are required to obtain theoptimally sharp melting curves. They also show that the optical changesin this system are completely decoupled from the melting properties(i.e., small aggregates can give sharp transitions but still not changecolor).

Example 21 Electrical Properties of Gold Nanoparticle Assemblies

Electron transport through DNA has been one of the most intenselydebated subjects in chemistry over the past five years. (Kelley et al.,Science 283:375–381 (1999); Turro et al., JBIC 3:201–209 (1998); Lewiset al., JBIC 3:215–221 (1998); Ratner, M. Nature 397:480–481 (1999);Okahata et al., J. Am. Chem. Soc. 120:6165–6166 (1998)) Some claim thatDNA is able to efficiently transport electrons, while others believe itto be an insulator.

In a seemingly disparate field of study, a great deal of effort has beendevoted to examining the electrical properties of nanoparticle-basedmaterials. (Terrill et al., J. Am. Chem. Soc. 117:12537–12548 (1995);Brust et al., Adv. Mater. 7:795–797 (1995); Bethell et al., J.Electroanal. Chem. 409:137–143 (1996); Musick et al., Chem. Mater.9:1499–1501 (1997); Brust et al., Langmuir 14:5425–5429 (1998); Collieret al., Science 277:1978–1981 (1997)). Indeed, many groups have exploredways to assemble nanoparticles into two- and three-dimensional networksand have investigated the electronic properties of such structures.However, virtually nothing is known about the electrical properties ofnanoparticle-based materials linked with DNA.

For the first time, in this study, the electrical properties of goldnanoparticle assemblies, formed with different length DNA interconnectshave been examined. As shown below, these hybrid inorganic assembliesbehave as semiconductors, regardless of oligonucleotide particleinterconnect length over a 24 to 72 nucleotide range. The resultsreported herein indicate that DNA interconnects can be used aschemically specific scaffolding materials for metallic nanoparticleswithout forming insulating barriers between them and thereby destroyingtheir electrical properties. These results point towards new ways suchhybrid assemblies can be exploited as electronic materials.

At the heart of this issue is the following question: Can nanoparticlesassembled by DNA still conduct electricity or will the DNAinterconnects, which are heavily loaded on each particle, (Mucic, R. C.Synthetically Programmable Nanoparticle Assembly Using DNA, Thesis Ph.D., Northwestern University (1999)) act as insulating shells? Theconductivities of these materials as a function of temperature,oligonucleotide length, and relative humidity were examined. TheDNA-linked nanoparticle structures were characterized by field emissionscanning electron microscopy (FE-SEM), synchrotron small angle x-rayscattering (SAXS) experiments, thermal denaturation profiles, and UV-visspectroscopy.

In a typical experiment (see FIG. 40), citrate-stabilized 13 nm goldnanoparticles were modified with 3′ and 5′ alkanethiol-capped 12-meroligonucleotides 1 (3′SH(CH₂)₃O(PO²⁻)O-ATGCTCAACTCT 5′ [SEQ ID NO:59])and 2 (5′SH(CH₂)₆O(PO²⁻)O-CGCATTCAGGAT 3′ [SEQ ID NO:50]) as describedin Examples 1 and 3. DNA strands with lengths of 24, 48, or 72 bases(3(5′TACGAGTTGAGAATCCTGAATGCG3′[SEQ ID NO:60]), 4(5′TACGAGTTGAGACCGTTAAGACGAGGCAATC-ATGCAATCCTGAATGCG 3′[SEQ ID NO:61]),and 5(5′TACGAGTTGAGACCGTTAAGACGAGGCAATCATGCATATATTGGACGCTTTACGGACAACATCCTGAATGCG3′[SEQ ID NO:62]) were used as linkers. Fillers 6(3′GGCAATTCTGCTCCGTTAGTACGT5′ [SEQ ID NO:63]) and 7(3′GGCAATTCTGCTCCGTTAGTACGTATATAACCTGCGAAATGCCTGTTG5′ [SEQ IN NO:64])were used with the 48 and 72 base linkers. The DNA-modifiednanoparticles and DNA linkers and fillers were stored in 0.3 M NaCl, 10mM phosphate (pH 7) buffer (referred as to 0.3 M PBS) prior to use. Toconstruct nanoparticle assemblies, 1-modified gold nanoparticles (652μl, 9.7 nM) and 2-modified gold nanoparticles (652 μl, 9.7 nM) wereadded to linker DNA 3, 4, or 5 (30 μl, 10 nM). After full precipitation,the aggregates were washed with 0.3 M CH₃COONH₄ solution to removeexcess linker DNA and NaCl.

Lyophilization (10⁻³˜10⁻² torr) of the aggregate to dryness results inpellets and removal of the volatile salt, CH₃COONH₄. Unfunctionalized,citrate-stabilized particles, prepared by the Frens method, (Frens,Nature Phys. Sci. 241:20–22 (1973)) were dried as a film and used forcomparison purposes. The resulting dried aggregates had a colorresembling tarnished brass and were very brittle. FE-SEM imagesdemonstrated that oligonucleotide-modified nanoparticles remained intactupon drying, while citrate-stabilized nanoparticles fused to oneanother. Significantly, the dried DNA-linked aggregates could beredispersed in 0.3 M PBS buffer (1 ml), and exhibited excellent meltingproperties; heating such a dispersion to 60° C. resulted indehybridization of the DNA interconnects, yielding a red solution ofdispersed nanoparticles. This combined with the FE-SEM data conclusivelydemonstrated that DNA-modified gold nanoparticles are not irreversiblyaggregated upon drying.

The electrical conductivities of the three samples (dried aggregateslinked by 3,4, and 5, respectively) were measured using acomputer-controlled, four-probe technique. Electrical contacts consistedof fine gold wires (25 and 60 μm diameter) attached to pellets with goldpaste. Samples were cooled in a moderate vacuum (10⁻³ to 10⁻² torr), andconductivity was measured as the temperature was increased under a dry,low pressure of helium gas. The sample chamber was insulated from lightin order to eliminate possible optoelectronic effects. Excitationcurrents were kept at or below 100 nA, and the voltage across the entiresample was limited to a maximum of 20 V. Surprisingly, theconductivities of the aggregates formed from all three linkers, rangedfrom 10⁻⁵ to 10⁻⁴ S/cm at room temperature, and they showed similartemperature dependent behavior. The conductivities of the DNA-linkedaggregates showed Arrhenius behavior up to about 190° K., which ischaracteristic of a semiconducting material. This is similar to thebehavior of activated electron hopping observed in discontinuous metalisland films (Barwinski, Thin Solid Films 128:1–9 (1985)). Goldnanoparticle networks linked by alkanedithiols have shown similartemperature dependence (Brust et al., Adv. Mater. 7:795–797 (1995);Bethell et al., J. Electroanal. Chem. 409:137–143 (1996)). Activationenergies of charge transport can be obtained from a plot of ln σ versus1/T using equation (1).σ=σ_(o)exp[−E _(a)/(kT)]  (1)The average activation energies calculated from three measurements were7.4±0.2 meV, 7.5±0.3 meV, and 7.6±0.4 meV for the 24-, 48-, and 72-merlinkers, respectively. Conductivity data from 50° K. to 150° K. wereused for these calculations.

Since the electrical properties of these types of materials shoulddepend on the distance between particles, synchrotron SAXS experimentswere used to determine interparticle distances of the dispersed anddried aggregates. The SAXS experiments were performed at theDupont-Northwestern-Dow Collaborative Access Team (DND-CAT) Sector 5 ofthe Advanced Photon Source, Argonne National Laboratory. DNA-linkedaggregates and dilute samples of DNA-modified colloid were irradiatedwith an 0.3 micron beam of 1.54 Å radiation, and scattered radiation wascollected on a CCD detector. The 2D data were circularly averaged andtransformed into a function, I (s), of the scattering vector magnitude,s=2sin(θ)/λ, where 2θ is the scattering angle and λ is the wavelength ofthe incident radiation. All data were corrected for backgroundscattering and sample absorption. The first peak position, which issensitive to interparticle distance, drastically changed from s valuesof 0.063 nm⁻¹, 0.048 nm⁻¹, and 0.037 nm⁻¹ for the 24-, 48-, and 72-merlinked aggregates, respectively, to an s value of 0.087 nm⁻¹ upon dryingfor all three aggregates structures. This indicates that interparticledistances decreased significantly upon drying, to the point where theparticles were almost touching, and that such distances were virtuallyindependent of linker length, while those in solution were highlydependent on linker length. This explains why similar activationenergies were observed for the three different linker systems in thedried pellet conductivity experiments. Moreover, it also explains whyrelatively high conductivities were observed, regardless of how oneviews the electronic properties of DNA. Unlike the DNA-linked materials,the dried film of citrate-stabilized gold nanoparticles showed metallicbehavior. This is consistent with the SEM data, which showed that suchparticles fuse together.

Above 190° K., the measured conductivities of the DNA-linked samplesshowed an anomalous dipping behavior. For all samples, the conductivitystarted to decrease abruptly at approximately 190° K. and continued todecrease until approximately 250° K., at which point it increased again.To investigate this unusual behavior in detail, the electricalconductivity was measured as the sample was cooled and warmedrepeatedly. Interestingly, the dip in conductivity only occurred in thedirection of increasing temperature. Since DNA is hydrophilic and watercould potentially affect the electrical properties of the hybridstructures, the effect of relative humidity on the conductivity of thegold aggregates was examined. The resistance increased by a factor of 10with increasing humidity from 1% to 100%. It should be noted that thecharacteristic dip was very weak when the sample was kept in vacuum(10⁻⁶ Torr) for 48 hours prior to the conductivity measurement. Fromthese observations, it was concluded that the unusual dip and subsequentrise in conductivity above 190° K. is associated with water melting andthe hygroscopic nature of the DNA, which temporarily increased theinterparticle distance (until evaporation took place). Consistent withthis hypothesis, SAXS measurements on a dried aggregate that was wettedwith 0.3 M PBS buffer showed a 200% increase in interparticle distance(˜2 nm).

These studies are important for the following reasons. First, they showthat one can use the molecular recognition properties of DNA to assemblenanoparticle-based materials without passivating them or destroyingtheir discrete structural or electrical properties. If theseDNA-functionalized particles are to be used to study electricaltransport in three-dimensional macroscopic assemblies or evenlithographically patterned structures (Piner et al., Science 283:661–663(1999)), it is imperative that their electrical transport properties bedelineated. Second, it shows that over a fairly long linker distance(8–24 nm), the conductivities of the dried assemblies are virtuallyindependent of DNA linker length. This is likely a result of the removalof water and the use of a volatile salt in these experiments; indeed,the free volume created by removal of solvent and salt allows the DNA tobe compressed on the surface and close approach of the particles withinthe aggregates. Third, the aggregates with the DNA-protectednanoparticles behave as semiconductors, while films formed fromcitrate-stabilized particles exhibit irreversible particle fusion andmetallic behavior. Finally, these results point toward the use of thesematerials in DNA diagnostic applications where sequence specific bindingevents between nanoparticles functionalized with oligonucleotides andtarget DNA effect the closing of a circuit and a dramatic increase inconductivity (i.e. from an insulator to a semiconductor) (see nextexample).

Example 22 Detection of Nucleic Acid Using Gold Electrodes

A method of detecting nucleic acid using gold electrodes is illustrateddiagramatically in FIG. 41. A glass surface between two gold electrodeswas modified with 12-mer oligonucleotides 1 (3′NH₂(CH₂)₇O(PO²⁻)O-ATG-CTC-AAC-TCT [SEQ ID NO:59]) complementary totarget DNA 3 (5′ TAC GAG TTG AGA ATC CTG AAT GCG [SEQ ID NO:60]) by themethod of Guo at al., Nucleic Acids Res., 22, 5456–5465 (1994).Oligonucleotides 2 (5′ SH(CH₂)₆O(PO²⁻)O-CGC-ATT-CAG-GAT [SEQ ID NO:50])were prepared and attached to 13 nm gold nanoparticles as described inExamples 1 and 18 to yield nanoparticles a. Target DNA 3 andnanoparticles a were added to the device. The color of the glass surfaceturned pink, indicating that target DNA-gold nanoparticle assemblieswere formed on the glass substrate. Next, the device was immersed in 0.3M NaCl, 10 mM phosphate buffer and heated at 40° C. for 1 hour to removenonspecifically bound DNA, and then treated with a silver stainingsolution as described in Example 19 for 5 minutes. The resistance of theelectrode was 67 kΩ.

For comparison, a control device modified by attaching oligonucleotides4, instead of oligonucleotides 1, between the electrodes.Oligonucleotides 4 have the same sequence (5′NH₂(CH₂)₆O(PO²⁻)O-CGC-ATT-CAG-GAT [SEQ ID NO:50]) as oligonucleotides 2on the nanoparticles and will bind to target DNA 3 so as to preventbinding of the nanoparticles. The test was otherwise performed asdescribed above. The resistance was higher than 40 MΩ, the detectionlimit of the multimeter that was used.

This experiment shows that only complementary target DNA strands formnanoparticle assemblies between the two electrodes of the device, andthat the circuit can be completed by nanoparticle hybridization andsubsequent silver staining. Therefore, complementary DNA andnoncomplementary DNA can be differentiated by measuring conductivity.This format is extendable to substrate arrays (chips) with thousands ofpairs of electrodes capable of testing for thousands of differentnucleic acids simultaneously.

1. A satellite probe comprising: a particle having attached theretooligonucleotides, the oligonucleotides having a sequence wherein atleast a portion of the sequence of the oligonucleotides is complementaryto at least a portion of a sequence of a nucleic acid target; and probeoligonucleotides hybridized to the oligonucleotides attached to theparticles, the probe oligonucleotides having a sequence wherein at leasta portion of the sequence of the probe oligonucleotides is complementaryto at least a portion of the sequence of the oligonucleotides attachedto the particles and identical to at least a portion of the sequence ofthe nucleic acid target; and a reporter attached each probeoligonucleotide.
 2. The satellite probe of claim 1 wherein the particlesare magnetic.
 3. The satellite probe of claim 1 wherein the reportermolecule is a fluorescent molecule.
 4. The satellite probe of claim 1wherein the reporter molecule is a dye molecule.
 5. The satellite probeof claim 1 wherein the reporter molecule is a redox-active molecule. 6.The satellite probe of claim 1 wherein the particles are modified with amaterial capable of quenching fluorescence of fluorescent moleculesattached to the probe oligonucleotides.
 7. The satellite probe of claim1 wherein the material is a dye.
 8. The satellite probe of claim 5wherein the particles are modified with dye molecules having opticalproperties that are distinct from the reporter molecule.
 9. Thesatellite probe of claim 1 wherein the redox-active molecule is aferrocene derivative.
 10. The satellite probe of claim 1 wherein theparticles are nanoparticles.
 11. The satellite probe of claim 10 whereinthe oligonucleotides are present on surface of the nanoparticles at asurface density of at least 10 picomoles/cm².
 12. The satellite probe ofclaim 11 wherein the oligonucleotides are present on surface of thenanoparticles at a surface density of at least 15 picomoles/cm².
 13. Thesatellite probe of claim 12 wherein the oligonucleotides are present onsurface of the nanoparticles at a surface density from about 15picomoles/cm² to about 40 picomoles/cm².
 14. The satellite probe ofclaim 10 wherein the nanoparticles are metal nanoparticles orsemiconductor nanoparticles.
 15. The satellite probe of claim 10 whereinthe nanoparticles are gold nanoparticles.
 16. The satellite probe ofclaim 1 wherein at least some of the oligonucleotides on each type ofparticles comprise at least one type of recognition oligonucleotides,each type of recognition oligonucleotides comprising a spacer portionand a recognition portion, the spacer portion being designed so that itis bound to the particles, the recognition portion having a sequencecomplementary to at least one portion of a sequence of a nucleic acidtarget.
 17. The satellite probe of claim 16 wherein the spacer portionhas a moiety covalently bound to it, the moiety comprising a functionalgroup through which the spacer portion is bound to the particles. 18.The satellite probe of claim 16 wherein the spacer portion comprises atleast about 10 nucleotides.
 19. The satellite probe of claim 18 whereinthe spacer portion comprises from about 10 to about 30 nucleotides. 20.The satellite probe of claim 16 wherein the bases of the nucleotides ofthe spacer portion are all adenines, all thymines, all cytosines, alluracils or all guanines.
 21. The satellite probe of claim 1 wherein atleast some the oligonucleotides on each type of particles comprise atleast one type of recognition oligonucleotides, each type of recognitionoligonucleotides comprising a sequence complementary to at least oneportion of a sequence of a nucleic acid target; and a type of diluentoligonucleotide.
 22. The satellite probe of claim 21 wherein, each typeof recognition oligonucleotides comprises a spacer portion and arecognition portion, the spacer portion being designed so that it isbound to the particles, the recognition portion having a sequencecomplementary to at least one portion of a sequence of a nucleic acidtarget or another oligonucleotide.
 23. The satellite probe of claim 22wherein the spacer portion has a moiety covalently bound to it, themoiety comprising a functional group through which the spacer portion isbound to the particles.
 24. The satellite probe of claim 22 wherein thespacer portion comprises at least about 10 nucleotides.
 25. Thesatellite probe of claim 24 wherein the spacer portion comprises fromabout 10 to about 30 nucleotides.
 26. The satellite probe of claim 22wherein the bases of the nucleotides of the spacer portion are alladenines, all thymines, all cytosines, all uracils or all guanines. 27.The satellite probe of claim 22 wherein the diluent oligonucleotidescontain about the same number of nucleotides as are contained in thespacer portions of the recognition oligonucleotides.
 28. The satelliteprobe of claim 27 wherein the sequence of the diluent oligonucleotidesis the same as that of the spacer portions of the recognitionoligonucleotides.
 29. The satellite probe of claim 5 wherein theoligonucleotides are attached to the nanoparticles in an agingprocessing comprising contacting the oligonucleotides with thenanoparticle in an aqueous solution for a period of time sufficient toallow some of the oligonucleotides to bind to the nanoparticle; addingat least one salt to the aqueous solution to form an aqueous saltsolution; and contacting the oligonucleotides and nanoparticle in anaqueous salt solution for an additional period of time sufficient toenable additional oligonucleotides to bind to the nanoparticle.
 30. Thesatellite probe of claim 29 wherein the oligonucleotides andnanoparticles are contacted in aqueous solution for about 12 to about 24hours.
 31. The satellite probe of claim 29 wherein salt is added to theaqueous solution to form the aqueous salt solution which is buffered atpH 7.0 and which contains about 0.1 M NaCl.
 32. The satellite probe ofclaim 29 wherein the oligonucleotides and nanoparticles are contacted inthe aqueous salt solution for an additional 40 hours to increase thedensity of oligonucleotides bound to the nanoparticles.
 33. Thesatellite probe of claim 31 wherein the salt is added to the aqueoussolution in a single addition.
 34. The satellite probe of claim 31wherein the salt is added gradually to the aqueous solution over time.35. The satellite probe of claim 31 wherein the salt is selected fromthe group consisting of sodium chloride, magnesium chloride, potassiumchloride, ammonium chloride, sodium acetate, ammonium acetate, acombination of two or more of these salts, one of these salts in aphosphate buffer, and a combination of two or more these salts in aphosphate buffer.
 36. The satellite probe of claim 35 wherein the saltis sodium chloride in a phosphate buffer.
 37. The satellite probe ofclaim 1 wherein the oligonucleotides are bound to the nanoparticlesthrough sulfur linkages.
 38. A kit comprising the satellite probe ofclaim 1.